Natural human antibody

ABSTRACT

A reshaped human anti-HM1.24 antibody comprising:
         (A) L chains each comprising (1) a constant region of a human L chain, and (2) FRs of a human L chain, and CDRs of L chain of mouse anti-HM1.24 monoclonal antibody; and   (B) H chains each comprising (2) a constant region of a human H chain, and (2) FRs of a human H chain, and CDRs of H chain of mouse anti-HM1.24 monoclonal antibody. Since the majority of the reshaped human antibody is derived from human antibody and the CDR has a low antigenicity, the reshaped human antibody of the present invention has low antigenicity and therefore is very promising in medical and therapeutic applications.

This application is a 35 U.S.C. §371 filing of PCT/J98/04469, filed onOct. 2, 1998.

TECHNICAL FIELD

The present invention relates to a method of preparing natural humanizedantibody and the natural humanized antibody obtained by said method ofpreparation. The present invention also relates to DNA encoding naturalhumanized antibody, an expression vector comprising said DNA, a hostcomprising said DNA, and a method of preparing natural humanizedantibody from cells into which said DNA has been introduced.

BACKGROUND ART

Mouse monoclonal antibodies can be relatively easily isolated by thewidely used hybridoma technology (Kohler, G. and Milstein, C. Nature(1975) 256, 495–497). On the other hand, a similar technique for humanhybridoma has yet to be widespread though it is expected to become so.Furthermore, there is a need for antibodies to human antigens inclinical applications, and therefore the generation of mouse monoclonalantibodies is indispensable for the development of antibodypharmaceuticals.

In fact, a number of monoclonal antibodies have been isolated againsttumor cells and viruses, and have been studied in clinical applications.It has been revealed, however, that mouse antibodies, which are aforeign substances to humans, induce HAMA (human anti-mouse antibody)due to the potent antigenicity, and that it is extremely unsuitable forclinical applications because of such problems as a weak activity ofinducing ADCC (Schroff, R. W., Cancer Res. (1985) 45, 879–885; Shawler,D. L., et al, J. Immunol. (1985) 135, 1530–1535).

In order to solve this problem, chimeric antibody was created(Neuberger, M. S. et al., Nature (1984) 312, 604–608; Boulianne, G. L.et al., Nature (1984) 312, 643–646). Chimeric antibody is made bylinking a variable region of a mouse antibody to a constant region of ahuman antibody, i.e. in chimeric antibody the constant region of themouse antibody which is responsible for a particularly potentantigenicity has been replaced with a human counterpart. This isexpected to enable a physiological binding with a human Fc receptor andto induce Fc-mediated functions. In fact, marked decreases inantigenicity has been reported in a clinical study using chimericantibodies (LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. U.S.A. (1989)86, 4220–4224). However, trouble-causing cases were reported thatdeveloped HAMA against mouse variable regions (LoBuglio, A. F. et al.,Proc. Natl. Acad. Sci. U.S.A. (1989) 86, 4220–4224).

Accordingly, methods have been developed, though more complicated, formaking a humanized antibody which is closer to a human antibody. This isa technique of reconstructing the antigen binding site of a mouseantibody on a human antibody (Jones, P. T. et al., Nature (1986) 321,5225–525; Verhoeyen, M. et al., Scinece (1988) 239, 1534–1536;Riechmann, L. et al., Nature (1988) 332,323–327)). Thus, a variableregion of an antibody, for both of the H chain and the L chain,comprises four framework regions (FRS) and three complementaritydetermining regions (CDRs) sandwiched between them.

It is known that CDR is mainly responsible for the formation of antigenbinding sites and some amino acid residues on the FR are involvedtherein either directly or indirectly. Since the basic structures ofantibodies are similar to each other, it was thought possible to graftan antigen binding site of an antibody to another antibody. The researchgroup led by G. Winter has, in fact, successfully grafted CDRs of amouse anti-rhizobium antibody to a human antibody (CDR-grafting) therebyobtaining a humanized antibody having a rhizobium binding activity(Jones, P. T. et al., Nature (1986) 321, 522–525).

In some cases, however, humanization by CDR-grafting alone does notprovide humanized antibody that has an antigen binding activity similarto the original mouse antibody. Accordingly, as described above,attempts have been made to replace some FR amino acid residues. FR aminoacid residues to be replaced are involved in the maintenance of thestructure of amino acid residues that constitute the basic structure ofan antibody molecule (canonical structure; Chothia, C. et al., Nature(1989) 342, 877–883; Chothia, C. and Lesk, A. M. J. Molec. Biol. (1987)196, 901–917) or CDRs, or directly interact with antigen molecules.

In fact, amino acid substitution on the FR has been made for most of thehumanized antibody, wherein artificial FR sequences that do notnaturally occur are formed. At times, too many amino acid substitutionshave been made, which makes doubtful the original meaning ofCDR-grafting for minimizing the antigenicity of mouse antibody (Queen,C. et al., Proc. Natl. Acad. Sci. U.S.A. (1989) 86, 10029–10033; Co, M.S. et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88, 2869–2873).

A solution to this problem is to devise methods of selecting human FRS.Thus, the number of FR amino acid residues to be replaced depends on thehomology between the FRs of the human antibody selected for CDR-graftingand the FRs of the original mouse antibody. Accordingly, human FRshaving a high homology with mouse FRs are usually selected so as tominimize the degree of substitution. However, in many cases even the FRsof humanized antibody thus obtained have amino acid sequences that donot occur naturally, which may present the problem of antigenicity.Thus, there is a need for the technology of constructing humanizedantibody that can solve the above problems, have lower probability ofinducing antigenicity, and have higher safety.

DISCLOSURE OF THE INVENTION

The present invention is an improvement of the conventional method ofconstructing humanized antibody, and provides a method of constructinghumanized antibody that completely retains the antigen binding activityof the original mouse antibody and that comprises naturally occurringhuman FRs, in other words a method of constructing humanized antibodythat involves no amino acid substitution on the FR.

Thus, the present invention provides a method of preparing a naturalhumanized antibody which comprises conducting a homology search for theFR of a primary design antibody and selecting a natural human FRretaining the artificial amino acid residues contained in the FR of theprimary design antibody and having a homology therewith. As used herein,the primary design antibody is a humanized antibody (also called areshaped human antibody) prepared by the conventional CDR-grafting.

The present invention also provides a method of preparing a naturalhumanized antibody which comprises conducting a homology search for theFR of a primary design antibody, selecting a natural human FR retainingthe artificial amino acid residues contained in the FR of the primarydesign antibody and having a homology therewith, and exchanging one or aplurality of different amino acid residues between the FR of the primarydesign antibody and the selected natural human FR.

Preferably, in the above method of preparation, the primary designantibody comprises the CDRs derived from a first animal species and theFRs derived from a second animal species. More preferably, in theprimary design antibody the first animal species is a non-human mammaland the second animal species is human. Examples of the first animalspecies, i.e. a mammal, include mouse, rat, hamster, rabbit, and monkey.

The present invention also provides a method of preparing a naturalhumanized antibody which comprises conducting a homology search for theFR of a primary design antibody, selecting a natural human FR retainingthe artificial amino acid residues derived from the FR of a non-humanantibody contained in the FR of the primary design antibody and having ahigh homology therewith, and exchanging one or a plurality of differentamino acid residues between the FR of the primary design antibody andthe selected natural human FR.

The present invention also provides a natural humanized antibodyobtained by the above preparation method.

The present invention also provides a natural humanized antibodycontaining the CDRs derived from a first animal species and the FRSderived from a second animal species characterized in that said FRscomprise an amino acid sequence which is different from the FRs used forCDR-grafting by one or a plurality of amino acid residues and isreplaced with the FR derived from the second animal species having thesame amino acid residues as said different amino acid residues at thesame positions. Preferably the first animal species is a non-humanmammal and the second animal species is human. Examples of the firstanimal species, i.e. a mammal, include mouse, rat, hamster, rabbit, andmonkey.

The present invention also provides DNA encoding the above naturalhumanized antibody.

The present invention also provides an expression vector comprising theabove DNA.

The present invention also provides a host comprising the above DNA.

The present invention also provides a method of preparing a naturalhumanized antibody which comprises culturing cells into which anexpression vector comprising the above DNA has been introduced andcollecting the desired natural humanized antibody from the culture ofsaid cells.

The present invention also provides a pharmaceutical compositioncomprising a natural humanized antibody.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a graph showing that the fluorescent intensity of chimericanti-HM1.24 antibody is shifted similarly to that of mouse anti-HM1.24antibody as compared to control antibody in the FCM analysis using ahuman myeloma cell line KPMM2.

FIG. 2 is a graph showing that chimeric anti-HM1.24 antibody inhibitsthe binding of biotinylated mouse anti-HM1.24 antibody to the WISH cellsin a dose-dependent manner similarly to that of mouse anti-HM1.24antibody.

FIG. 3 is a graph showing that chimeric anti-HM1.24 antibody has anincreased cytotoxic activity to the RPMI 8226 cells with increasing E/Tratios whereas control IgG1 or mouse anti-HM1.24 antibody has nocytotoxic activity to the RPMI 8226 cells.

FIG. 4 is a diagram showing a method of constructing the L chain ofreshaped human anti-HM1.24 antibody by CDR-grafting using the PCRmethod.

FIG. 5 is a diagram showing a method of constructing the H chain ofreshaped human anti-HM1.24 antibody in which oligonucleotides RVH1,RVH2, RVH3, and RVH4 are assembled by the PCR method.

FIG. 6 is a diagram showing a method of constructing the H chain Vregion of human-mouse hybrid anti-HM1.24 antibody.

FIG. 7 is a diagram showing a method of constructing the H chain Vregion of mouse-human hybrid anti-HM1.24 antibody.

FIG. 8 is a graph showing that the L chain version a of reshaped humananti-HM1.24 antibody has an antigen binding activity of a similar degreeto that of chimeric anti-HM1.24 antibody. In the figure, −1 and −2represent different lots.

FIG. 9 is a graph showing the antigen binding activity of reshaped humananti-HM1.24 antibody prepared from a combination of the L chain versiona and the H chain version a, b, f, or h, and chimeric anti-HM1.24antibody.

FIG. 10 is a graph showing the antigen binding activity of reshapedhuman anti-HM1.24 antibody prepared from a combination of the L chainversion b and the H chain version a, b, f, or h, and chimericanti-HM1.24 antibody.

FIG. 11 is a graph showing the binding inhibition activity of reshapedhuman anti-HM1.24 antibody prepared from a combination of the L chainversion a and the H chain version a, b, f, or h, and chimericanti-HM1.24 antibody.

FIG. 12 is a graph showing the binding inhibition activity of reshapedhuman anti-HM1.24 antibody prepared from a combination of the L chainversion b and the H chain version a, b, f, or h, and chimericanti-HM1.24 antibody.

FIG. 13 is a graph showing the antigen binding activity of the H chainversions a, b, c, and d of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 14 is a graph showing the antigen binding activity of the H chainversions a and e of reshaped human anti-HM1.24 antibody and chimericanti-HM1.24 antibody. In the figure, −1 and −2 represent different lots.

FIG. 15 is a graph showing the binding inhibition activity of the Hchain versions a, c, p, and r of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 16 is a graph showing the antigen binding activity of human-mousehybrid anti-HM1.24 antibody, mouse-human hybrid anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 17 is a graph showing the antigen binding activity of the H chainversion a, b, c, and f of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 18 is a graph showing the antigen binding activity of the H chainversions a and g of reshaped human anti-HM1.24 antibody and chimericanti-HM1.24 antibody.

FIG. 19 is a graph showing the binding inhibition activity of the Hchain versions a and g of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 20 is a graph showing the antigen binding activity of the H chainversions h and i of reshaped human anti-HM1.24 antibody and chimericanti-HM1.24 antibody.

FIG. 21 is a graph showing the antigen binding activity of the H chainversions f, h, and j of reshaped human anti-HM1.24 antibody and chimericanti-HM1.24 antibody.

FIG. 22 is a graph showing the binding inhibition activity of the Hchain versions h and i of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 23 is a graph showing the binding inhibition activity of the Hchain versions f, h, and j of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 24 is a graph showing the antigen binding activity of the H chainversions h, k, l, m, n, and o of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 25 is a graph showing the antigen binding activity of the H chainversions a, h, p, and q of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 26 is a graph showing the binding inhibition activity of the Hchain versions h, k, l, m, n, and o of reshaped human anti-HM1.24antibody and chimeric anti-HM1.24 antibody to the WISH cells.

FIG. 27 is a graph showing the binding inhibition activity of the Hchain versions a, h, p, and q of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 28 is a graph showing the antigen binding activity of the H chainversions a, c, p, and r of reshaped human anti-HM1.24 antibody andchimeric anti-HM1.24 antibody.

FIG. 29 is a graph showing that natural humanized anti-HM1.24 antibody(the secondary design antibody) has an antigen binding activity of asimilar degree to that of reshaped human anti-HM1.24 antibody (theprimary design antibody).

FIG. 30 is a graph showing that natural humanized anti-HM1.24 antibody(the secondary design antibody) has a binding inhibition activity of asimilar degree to that of reshaped human anti-HM1.24 antibody (theprimary design antibody).

FIG. 31 is a graph showing that purified reshaped human anti-HM1.24antibody has an antigen binding activity of a similar degree to that ofchimeric human anti-HM1.24 antibody.

FIG. 32 is a graph showing that purified reshaped human anti-HM1.24antibody has an binding inhibition activity of a similar degree to thatof chimeric human anti-HM1.24 antibody.

FIG. 33 is a graph showing that natural humanized anti-HM1.24 antibody(the secondary design antibody) has an increased cytotoxic activity tothe KPMM2 cells with increasing E/T ratios.

EMBODIMENT FOR CARRYING OUT THE INVENTION

1. Natural FR Sequence

In order to produce antibodies to a variety of antigens from the genescomprising limited antibody variable regions, organisms have a mechanismof introducing random gene mutations (called somatic mutations) in theantibody variable regions. In theory this should form extremely diverseFR amino acid sequences, but in practice positions of amino acidresidues more prone to the introduction of mutations and the kinds ofamino acid residues appear to be limited to a certain degree asdetermined by structural analysis of many human antibody FRs for whichactual structures have been elucidated.

As used herein, the term FR refers to the FR that has been defined inKabat, E. A. et al., Sequence of Proteins of Immunological Interest(1991). Thus, in the H chain, FR1 is amino acids No. 1 to 30, FR2 isamino acids No. 36 to 49, FR3 is amino acids No. 66 to 94, and FR4 isamino acids No. 103 to 113. On the other hand, in the L chain FR1 isamino acids No. 1 to 23, FR2 is amino acids No. 35 to 49, FR3 is aminoacids No. 57 to 88, and FR4 is amino acids No. 98 to 107.

2. From Human FR to Natural Human FR

In many cases, humanized antibodies (also called reshaped humanantibody) produced by the conventional CDR-grafting method have FR aminoacid sequences that cannot be found in nature. However, because avariety of FR amino acid sequences have already been found by somaticmutation as mentioned above, it is possible that FRs having artificialamino acid residues created by humanization could be converted intohuman FRs that occur in nature.

The present invention is intended to create humanized antibodycomprising naturally occurring human FRs in stead of artificial FRs byfurther processing humanized antibody that was constructed by theconventional humanization technology. When humanized antibody thatunderwent amino acid substitution is subjected to homology search usinghuman antibody FRs and known databases such as Swiss Plot (proteinsequence database), GenBank (nucleic acid sequence database), PRF(protein sequence database) PIR (protein sequence database), and GenPept(translanted protein sequence from GenBank), human FRs having completelymatched amino acid sequences or human FRs having homology can be found.

In the former case, FR substitution was carried out when seen from thehuman FR that was used as the acceptor of CDR-grafting, in which aformed FR that had been presumed to be artificial is present in thenatural FR, which can be considered an acceptor, and therefore an FRthat underwent no FR substitution can be obtained. In the latter case,by focusing on the amino acid sequence of human FR having a highhomology with an artificial FR, it is possible to effect amino acidsubstitution in the artificial FR that results in returning to asuitable natural human antibody thereby causing a complete match withthe natural human FR. This procedure represents humanization onCDR-grafted antibodies.

Since homology search of amino acid sequences between human antibodiesis conducted in this case, it is possible to find a human FR thatbelongs to the same subgroup as the human FR used in CDR-grafting and tofind an amino acid sequence having an extremely high homology. Thus, anatural human FR, obtained for each FR, more than satisfies theconsensus sequence of the subgroup though it is derived from differentantibodies.

3. Natural-Sequence Humanized Antibody

The natural humanized antibody obtained in the present inventioncomprises human antibody FRs that have been recognized to occur innature. Though FR1 to FR4 are sometimes derived from differentantibodies, homology search between human antibodies permits theselection of the antibodies that only belong to the same subgroup asdescribed above. The FR structure of each antibody in the same subgrouphas a structure very similar to another, and in fact humanizedantibodies based on consensus sequences in the subgroup have beengenerated (Kettleborough, C. A. et al., Protein Engng. (1991) 4,773–783; Satoh, K. et al., Molec. Immun. (1994) 31, 371–381).

It is believed that in antibodies, as described above, extremely diverseamino acid sequences occur naturally through somatic mutation. Only someof the structures have been characterized at present. If the FR sequenceof the antibody obtained cannot be found in nature, it is not clearwhether the FR is present in nature or not. When antibodies areconsidered as pharmaceuticals, the construction of CDR-grafting antibodycomprising naturally occurring human FRs provides such an antibody thathas properties superior to the conventional humanized antibodies from aviewpoint of of the object of the present invention to reduceantigenicity.

4. Method of Constructing Novel Humanized Antibody

The present invention solves the problem associated with humanizedantibody constructed by the conventional technique of humanization, thatis, it eliminates antigenicity arising from artificial FRs that are notfound in nature. Otherwise it is a technology to construct humanizedantibody by CDR-grafting composed of human FRs actually found in nature.The amino acid sequences of artificial FRs refer to the amino acidsequences of the FRs which as a whole cannot be found in nature. Theartificial amino acid sequences contained in FRs refer to those aminoacid sequences that cannot be found in nature in FRs.

As the amino acid sequences of FRs that are not found in nature, theremay be mentioned FRs having an amino acid sequence in which human aminoacid residues in a FR have returned to amino acid residues found in theFR of antibody derived from a non-human mammal which is a template ofhumanization in a humanized antibody constructed by the conventionalantibody-humanization technology. Alternatively, in a humanized antibodyconstructed by the conventional antibody-humanization technology, theremay be mentioned FRs having an amino acid sequence that are not found inthe antibodies derived from human and non-human mammals.

The method of producing the natural humanized antibody of the presentinvention is described hereinbelow.

First, a FR of the human antibody for use in CDR-grafting is selected bya conventional technique. The FR is subjected to amino acid substitutionto construct a humanized antibody having a biological activity equal toor higher than that of mouse antibody. This is considered as an endproduct of humanized antibody in the conventional method, but in thepresent invention it is a mere intermediate for construction of naturalhumanized antibody having a natural sequence. In the present inventionit is called the primary design antibody.

Subsequently, homology search is conducted on each of the FRs of theprimary design antibody. FRs having a complete match mean that the FRshave already comprised the natural FRs. On the other hand, a series ofnatural human FRs are listed that belong to the same subgroup as theprimary design antibody and having a homology but not a complete matchwith the primary design antibody. From the list, there may be selectedmost appropriate natural human FRS that maintain the amino acid residueof the FR derived from a non-human mammal such as mouse which wasimportant in the construction of the primary design antibody, and thathas a homology with the primary design antibody.

Homology search of FRS can be conducted using known databases. Examplesof such databases include Swiss Plot, GenBank, PRF, PIR, and GenPept.Homology search is conducted using these databases in which “the FRhaving a homology with the FR of the primary design antibody” listed byhomology search refers to the FR having a homology in the amino acidsequence of at least 80%, preferably at least 90%, more preferably atleast 91%, more preferably at least 92%, more preferably at least 93%,more preferably at least 94%, more preferably at least 95%, morepreferably at least 96% or greater, more preferably at least 97% orgreater, more preferably at least 98% or greater, and more preferably atleast 99% or greater. The homology of protein can be determined by thealgorithm described Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad.Sci. U.S.A. (1983) 80, 726–730.

Amino acid residues of a non-human mammal which were important forconstruction of the primary design antibody refers to the amino acidresidues derived from a non-human FR contained in an artificial FR. Manysuch amino acid residues are found in the amino acid residues (canonicalstructure) responsible for the basic structure of antibody molecule, theamino acid residues involved in the maintenance of the structure ofCDRs, or the amino acid residues that directly interact with antigenmolecule, and include for example an amino acid at position 71 of the Hchain, an amino acid at position 94 of the H chain, and the like, thoughthey may vary depending on the antibody.

As mentioned above, if one or a plurality of amino acid residuesdifferent between the FR of the primary design antibody and the naturalFR are replaced so as to produce humanized antibody having the aminoacid residues of a natural human FR, the humanized antibody (naturalhumanized antibody; termed the secondary design antibody) thus obtainedall comprise natural FRs. In this case all human FRs are preferablyhuman FRs that belong to the same subgroup, and more preferably arederived from the same antibody. Furthermore, all human FRs are notrequired to belong to the same subgroup, as long as they are reshapedinto an antibody and provide certain antigen binding activity, andthereby they are not limited to the human FRS that belong to the samesubgroup. According to the present invention, a plurality of amino acidresidues mean 2 or more amino acid residues, preferably 2 or more and 10or less amino acid residues, more preferably 2 or more and 5 or lessamino acid residues, more preferably 2 or more and 4 or less amino acidresidues, and more preferably 2 or more and 3 or less amino acidresidues in the amino acid sequence.

Homology between an artificial FR and a natural human FR is at least80%, preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, more preferably at least 93%, more preferablyat least 94%, more preferably at least 95%, more preferably at least 96%or greater, more preferably at least 97% or greater, more preferably atleast 98% or greater, and more preferably at least 99% or greater.

Then, the secondary design antibody is allowed to be expressed in asuitable expression system, for example in an animal cell, to evaluatethe antigen binding activity, and the like.

Furthermore, the method of preparation of the present invention can beeffected even without the actual construction of the primary designantibody. Thus, the primary design antibody is conventionally designed,and without the evaluation thereof the secondary design antibody may bedesigned, which may be directly evaluated. In fact, however, theidentification of important FR residues sometimes involves experiment,and the secondary design antibody is preferably constructed after theconventional primary design antibody has been experimentallyconstructed.

Specifically, in one aspect of the present invention, the naturalhumanized antibody of the present invention was produced with mouseanti-HM1.24 antibody (Goto, T. et al., Blood (1994) 84, 1922–1930) as atemplate.

For natural humanized antibodies designed as mentioned above, the geneencoding them can be obtained by a known method. For example, severaloligonucleotides are synthesized that have overlapping endscorresponding to the DNA encoding the amino acid sequence of thedesigned natural humanized antibody. A PCR method is carried out usingthese oligonucleotides as primers. Then, a PCR method is carried outusing primers that define the both ends of the DNA encoding the aminoacid sequence of the designed natural humanized antibody to obtain thegene encoding the desired natural humanized antibody.

Genes encoding a natural humanized antibody constructed as describedabove may be expressed in a known method so as to obtain the naturalhumanized antibody. In the case of mammalian cells, expression may beaccomplished using a commonly used useful promoter/enhancer, theantibody gene to be expressed, and DNA in which the poly A signal hasbeen operably linked at 3′ downstream thereof, or using a vectorcontaining the same. Examples of the promoter/enhancer include humancytomegalovirus immediate early promoter/enhancer.

Additionally, as the promoter/enhancer which can be used for expressionof antibody for use in the present invention, there can be used viralpromoters/enhancers such as retrovirus, polyoma virus, adenovirus, andsimian virus 40 (SV40), and promoters/enhancers derived from mammaliancells such as human elongation factor 1α (HEF1α).

For example, expression may be readily accomplished by the method ofMulligan et al. (Nature (1979) 277, 108) when the SV40 promoter/enhanceris used, or by the method of Mizushima et al. (Nucleic Acids Res. (1990)18, 5322) when the HEF1α promoter/enhancer is used.

In the case of Escherichia coli (E. coli), expression may be effected byoperably linking a commonly used useful promoter, a signal sequence forantibody secretion, and the antibody gene to be expressed, followed byexpression thereof. As the promoter, for example, there can be mentionedthe lacz promoter and the araB promoter. The method of Ward et al.(Nature (1098) 341, 544–546; FASEB J. (1992) 6, 2422–2427) may be usedwhen lacz promoter is used, and the method of Better et al. (Science(1988) 240, 1041–1043) may be used when araB promoter is used.

As the signal sequence for antibody secretion, when produced in theperiplasm of E. coli, the pelB signal sequence (Lei, S. P. et al., J.Bacteriol. (1987) 169, 4379) can be used. After separating the antibodyproduced in the periplasm, the structure of the antibody isappropriately refolded before use (see, for example, InternationalPatent Publication WO 96/30394, and Japanese Examined Patent Publication(Kokoku) No. 7(1995)-93879).

As the origin of replication, there can be used those derived from SV40,polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like.Furthermore, for the amplification of the gene copy number in the hostcell system, expression vectors can include as selectable markers theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, thedihydrofolate reductase (dhfr) gene and the like.

For the production of antibody for use in the present invention, anyproduction system can be used. The production system of antibodypreparation comprises the in vitro or the in vivo production system. Asthe in vitro production system, there can be mentioned a productionsystem which employs eukaryotic cells and the production system whichemploys prokaryotic cells.

When the eukaryotic cells are used, there are the production systemswhich employ animal cells, plant cells, and fungal cells. Known animalcells include (1) mammalian cells such as CHO cells (J. Exp. Med. (1995)108, 945), COS cells, myeloma cells, baby hamster kidney (BHK) cells,HeLa cells, and Vero cells, (2) amphibian cells such as Xenopus oosytes(Valle, et al., Nature (1981) 291, 358–340), or (3) insect cells such assf9, sf21, and Tn5. As CHO cells, preferably dhfr-CHO (Proc. Natl. Acad.Sci. U.S.A. (1980) 77, 4216–4220) that lacks the DHFR gene and CHO K-1(Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275) may be used.

Known plant cells include, for example, those derived from Nicotianatabacum, which is subjected to callus culture. Known fungal cellsinclude yeasts such as the genus Saccharomyces, for exampleSaccharomyces cereviceae, or filamentous fungi such as the genusAspergillus, for example Aspergillus niger.

When the prokaryotic cells are used, there are the production systemswhich employ bacterial cells. Known bacterial cells include Escherichiacoli (E. coli), and Bacillus subtilis.

By transforming these cells with the gene encoding the natural humanizedantibody of the present invention and and culturing the transformedcells in vitro, the natural humanized antibody can be obtained.Culturing is carried out in a known method. For example, as the cultureliquid, DMEM, MEM, RPMI1640, and IMDM can be used, and serum supplementssuch as fetal calf serum (FCS) may be used in combination, or serum-freeculture medium may be used. In addition, antibodies may be produced invivo by implanting cells into which the antibody gene has beenintroduced into the abdominal cavity of an animal and the like.

As in vivo production systems, there can be mentioned those which employanimals and those which employ plants. The gene of antibody isintroduced into an animal or a plant, and the antibody is produced insuch an animal or a plant and then collected.

When animals are used, there are the production systems which employmammals and insects.

As mammals, goats, pigs, sheep, mice, and cattle can be used (VickiGlaser, SPECTRUM Biotechnology Applications, 1993). When mammals areused, transgenic animals can also be used.

For example, an antibody gene is inserted into a gene encoding proteinwhich is inherently produced in the milk such as goat β casein toprepare fusion genes. DNA fragments containing the fusion gene intowhich the antibody gene has been inserted are injected into a goatembryo, and the embryo is introduced into a female goat. The desiredantibody is obtained from the milk produced by the transgenic goat borneto the goat who received the embryo or offsprings thereof. In order toincrease the amount of milk containing the desired antibody produced bythe transgenic goat, hormones may be given to the transgenic goat asappropriate (Ebert, K. M. et al., Bio/Technology (1994) 12, 699–702).

When insects are used, silkworms can be used. When silkworms are used,baculovirus into which the desired antibody gene has been inserted isinfected to the silkworm, and the desired antibody can be obtained fromthe body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315,592–594).

When plants are used, tabacco, for example, can be used. Moreover, whentabacco is used, the desired antibody gene is inserted into anexpression vector for plants, for example pMON 530, and then the vectoris introduced into a bacterium such as Agrobacterium tumefaciens. Thebacterium is then infected to tabacco such as Nicotiana tabacum toobtain the desired antibody from the leaves of the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131–138).

As described above, “hosts” as used herein encompasses animals andplants that produce the desired natural humanized antibody. Whenantibody is produced in vitro or in vivo production systems, asdescribed above, DNA encoding an H chain or an L chain of an antibodymay be separately integrated into an expression vector and a host istransformed simultaneously, or DNA encoding an H chain and DNA encodingan L chain may be integrated into a single expression vector and a hostis transformed therewith (see International Patent Publication WO94-11523).

As method of introducing an expression vector into a host, a knownmethod such as the calcium phosphate method (Virology (1973) 52,456–467) and the electropolation method (EMBO J. (982) 1, 841–845) andthe like can be used.

A natural humanized antibody produced and expressed as described abovecan be separated from the inside or outside of the cell or from the hostand then may be purified to homogeneity. Separation and purification ofthe natural humanized antibody for use in the present invention may beaccomplished by conventional methods of separation and purification usedfor protein, without any limitation. Separation and purification may beaccomplished by combining, as appropriate, chromatography such asaffinity chromatography, filtration, ultrafiltration, salting-out,dialysis and the like (Antibodies: A Laboratory Manual, Ed Harlow andDavid Lane, Cold Spring Harbor Laboratory, 1988).

As the column used for such affinity chromatography, there can bementioned Protein A column and Protein G column. As carriers for use inthe Protein A column there can be mentioned Hyper D, POROS, SepharoseF.F. (Pharmacis) and the like.

Chromatography other than affinity chromatography includes, for example,ion exchange chromatography, hydrophobic chromatography, gel-filtration,reverse-phase chromatography, adsorption chromatography and the like(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual. Ed Daniel R. Marshak et al., Cold Spring HarborLaboratory Press, 1996).

These chromatographies can be carried out using a liquid chromatographysuch as HPLC, FPLC, and the like.

The concentration of the natural humanized antibody of the presentinvention can be determined by the measurement of absorbance or by theenzyme-linked immunosorbent assay (ELISA) and the like. Thus, whenabsorbance measurement is employed, the natural humanized antibodyobtained is appropriately diluted with PBS and then the absorbance ismeasured at 280 nm, followed by calculation using the absorptioncoefficient of 1.35 OD at 1 mg/ml.

When the ELISA method is used, measurement is conducted as follows.Thus, 100 μl of goat anti-human IgG (manufactured by BIO SOURCE) dilutedto 1 mg/ml in 0.1 M bicarbonate buffer, pH 9.6, is added to a 96-wellplate (manufactured by Nunc), and is incubated overnight at 4° C. toimmobilize the antibody. After blocking, 100 μl each of appropriatelydiluted natural humanized antibody of the present invention or a samplecontaining the antibody, or human IgG (manufactured by CAPPEL) of aknown concentration as the standard is added, and incubated at roomtemperature for 1 hour.

After washing, 100 μl of 5000-fold diluted alkaline phosphatase-labeledanti-human IgG antibody (manufactured by BIO SOURCE) is added, andincubated at room temperature for 1 hour. After washing, the substratesolution is added and incubated, followed by the measurement ofabsorbance at 405 nm using the MICROPLATE READER Model 3550(manufactured by Bio-Rad) to calculate the concentration of the desiredantibody. BIAcore (manufactured by Pharmacia) can be used for themeasurement of antibody concentration.

The antigen binding activity, binding inhibition activity, andneutralizing activity of the natural humanized antibody of the presentinvention can be evaluated by known methods. For example, as methods ofdetermining the activity of the natural humanized antibody of thepresent invention, there can be mentioned ELISA, EIA(enzymeimmunoassay), RIA (radioimmunoassay), or fluorescent antibodymethod. For the evaluation of the above antibody, BIAcore (manufacturedby Pharmacia) can be used.

The natural humanized antibody of the present invention may be antibodyfragments or modified versions thereof. For example, as fragments ofantibody, there may be mentioned Fab, F(ab′)₂, Fv or single-chain Fv(scFv). scFv has a structure in which Fvs of the H chain and the L chainare ligated via a suitable linker.

In order to produce these antibodies, antibodies are treated with anenzyme such as papain or pepsin, or genes encoding these antibodyfragments are constructed and then introduced into an expression vector,which is expressed in a suitable host cell to express them (see, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2968–2976; Better, M.and Horwitz, A. H., Methods in Enzymology (1989) 178, 476–496, AcademicPress Inc.; Plucktrun, A. and Skerra, A., Methods in Enzymol. (1989)178, 476–496, Academic Press Inc.; Lamoyi, E., Methods in Enzymol.(1986) 121, 652–663; Rousseaux, J. et al., Methods in Enzymol. (1986)121, 663–669; Bird, R. E. and Walker, B. W., TIBTECH (1991) 9, 132–137).

scFv can be obtained by ligating the V region of H chain and the Vregion of L chain of antibody (see, International Patent Publication WO88-09344). In scFv, the V region of H chain and the V region of L chainare preferably ligated via a linker, preferably a peptide linker(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,5879–5883). The V region of H chain and the V region of L chain in thescFv may be derived from any of the above-mentioned antibodies. As thepeptide linker for ligating the V regions, any single-chain peptidecomprising, for example, one comprising 12 to 19 amino acid residues maybe used (see, U.S. Pat. No. 5,525,491).

DNA encoding scFv can be obtained using DNA encoding the H chain or theH chain V region of the above antibody and DNA encoding the L chain orthe L chain V region of the above antibody as the template by amplifyingthe portion of the DNA encoding the desired amino acid sequence amongthe above sequences by the PCR technique with the primer pair specifyingthe both ends thereof, and by further amplifying the combination of DNAencoding the peptide linker portion and the primer pair which definesthat both ends of said DNA be ligated to the H chain and the L chain,respectively.

Once DNAs encoding scFv are constructed, an expression vector containingthem and a host transformed with said expression vector can be obtainedby the conventional methods, and scFv can be obtained using theresultant host by the conventional methods.

These antibody fragments can be produced by obtaining the gene thereofin a similar manner to that mentioned above and by allowing it to beexpressed in a host. “Antibody” as used in the claim of the presentapplication encompasses these antibody fragments.

As modified antibodies, antibodies associated with various moleculessuch as polyethylene glycol (PEG) can be used. “Antibody” as used in theclaim of the present application encompasses these modified antibodies.These modified antibodies can be obtained by chemically modifying theantibodies thus obtained. These methods have already been established inthe art.

The natural humanized antibody of the present invention may beadministered orally or pareterally, either systemically or topically.The parenteral route may be selected from intravenous injection such asdrip infusion, intramuscular injection, intraperitoneal injection, andsubcutaneous injection, and the method of administration may be chosen,as appropriate, depending on the age and the condition of the patient.

The natural humanized antibody of the present invention may beadministered at a dosage that is sufficient to treat or to block atleast partially the pathological condition. For example, the effectivedosage is chosen from the range of 0.01 mg to 100 mg per kg of bodyweight per administration. Alternatively, the dosage in the range of 1to 1000 mg, preferably 5 to 50 mg per patient may be chosen. However,the natural humanized antibody of the present invention is not limitedto these dosages.

The natural humanized antibody of the present invention may containpharmaceutically acceptable carriers or additives depending on the routeof administration. Examples of such carriers or additives include water,a pharmaceutical acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-solubledextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethylcellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin,propylene glycol, polyethylene glycol, Vaseline, paraffin, stearylalcohol, searic acid, human serum albumin (HSA), mannitol, sorbitol,lactose, a pharmaceutically acceptable surfactant and the like.Additives used are chosen from, but not limited to, the above orcombinations thereof depending on the dosage form.

REFERENCE EXAMPLES

Before explaining the present invention with reference to the workingexamples, reference examples will be described as the premise thereof.

Reference Example 1 Cloning of cDNA Encoding the Variable Region of aMouse Anti-HM1.24 Antibody

1. Isolation of Messenger RNA (mRNA)

Using the Fast Track mRNA Isolation Kit Version 3.2 (manufactured byInvitrogen) according to the instruction attached thereto, mRNA wasisolated from 2×10⁸ hybridoma cells (FERM BP-5233) that produce a mouseanti-HM1.24 antibody.

2. Amplification of the Gene Encoding the Variable Region of Antibody bythe PCR Method

PCR was carried out using the amplification Thermal Cycler (manufacturedby Perkin Elmer Cetus).

2-1. Amplification and Fragmentation of the Gene Encoding the V Regionof a Mouse L Chain

From the mRNA thus isolated, single stranded cDNA was synthesized usingthe AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(manufactured by Life Science) and used for PCR. As primers used forPCR, MKV (Mouse Kappa Variable) primers (Jones, S. T. et al,Bio/Technology, 9, 88–89, (1991)) shown in SEQ ID NO: 29 to 39 thathybridize with the leader sequence of a mouse kappa type L chain wereused.

A hundred microliters of the PCR solution containing 10 mM Tris-HCl (pH8.3), 50 mM KCl, 0.1 mM dNTPS (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl2, 5units of DNA polymerase Ampli Taq (manufactured by Perkin Elmer Cetus),0.25 mM of the MKV primers shown in SEQ ID NO: 29 to 39, 3 mM of the MKCprimer shown in SEQ ID NO: 40, and 100 ng of single stranded cDNA wascovered with 50 μl of a mineral oil, and then heated at an initialtemperature of 94° C. for 3 minutes, and then at 94° C. for 1 minute, at55° C. for 1 minute, and at 72° C. for 1 minute in this order. Afterrepeating this cycle for 30 times, the reaction mixture was incubated at72° C. for 10 minutes. The amplified DNA fragment was purified by thelow melting point agarose (manufactured by Sigma), and digested withXmaI (manufactured by New England Biolabs) and SalI (manufactured byTakara Shuzo) at 37° C.

2-2. Amplification and Fragmentation of cDNA Encoding the V Region of aMouse H Chain

The gene encoding the V region of a mouse H chain was amplified by the5′-RACE method (Rapid Amplification of cDNA ends; Frohman, M. A. et al.,Proc. Natl. Acad. Sci. USA, 85, 8998–9002, (1988), Edwards, J. B. D. M.,et al., Nucleic Acids Res., 19, 5227–5232, (1991)). After cDNA wassynthesized using primer P1 (SEQ ID NO: 63) that specifically hybridizeswith the constant region of mouse IgG2a, cDNA encoding the V region of amouse H chain was amplified by the 5′-AmpliFINDER RACE KIT (manufacturedby CLONTECH) using the primer MHC 2a (SEQ ID NO: 64) that specificallyhybridizes with the constant region of mouse IgG2a and the anchor primer(SEQ ID NO: 101) attached to the kit. The amplified DNA fragment waspurified with the low melting point agarose (manufactured by Sigma) anddigested with EcoRI (manufactured by Takara) and XmaI (manufactured byNew England Biolabs) at 37° C.

3. Linking and Transformation

The DNA fragment comprising the gene encoding the V region of the mousekappa type L chain prepared as above was ligated to the pUC19 vectorprepared by digesting with SalI and XmaI by reacting in a reactionmixture containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 10 mMdithiothreitol, 1 mM ATP, 50 mg/ml of polyethylene glycol (8000) and oneunit of T4 DNA ligase (manufactured by GIBCO-BRL) at 16° C. for 2.5hours. Similarly, the gene encoding the V region of the mouse H chainwas reacted and ligated to pUC19 vector prepared by digesting with EcoRIand XmaI at 16° C. for three hours.

Then 10 μl of the above ligation mixture was added to 50 μl of thecompetent cells of Escherichia coli DH5, which was left on ice for 30minutes, at 42° C. for one minute, and again on ice for one minute.Subsequently 400 μl of 2×YT medium (Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989))was added thereto, incubated at 37° C. for one hour, and then the E.coli was plated on the 2×YT agar medium (Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989))containing 50 μg/ml of ampicillin, and then incubated overnight at 37°C. to obtain the E. coli transformant.

The transformant was cultured overnight at 37° C. in 10 ml of the 2×YTmedium containing 50 μg/ml of ampicillin, and then from this cultureplasmid DNA was prepared using the alkali method (Molecular Cloning: ALaboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press,(1989)).

The plasmid thus obtained containing the gene encoding the V region ofthe mouse kappa type L chain derived from the hybridoma that producesthe anti-HM1.24 antibody was termed pUCHMVL9. The plasmid obtained inthe above-mentioned method containing the gene encoding the V region ofthe mouse H chain derived from the hybridoma that produces theanti-HM1.24 antibody was termed pUCHMVHR16.

Reference Example 2 Determination of the Nucleotide Sequence of DNA

The nucleotide sequence of the cDNA coding region in the above-mentionedplasmid was determined using the automatic DNA sequencer (manufacturedby Applied Biosystem Inc.) and Taq Dye Deoxy Terminator Cycle SequencingKit (manufactured by Applied Biosystem Inc.) in the protocol indicatedby the manufacturer.

The nucleotide sequence of the gene encoding the V region of the L chainof the mouse anti-HM1.24 antibody contained in the plasmid pUCHMVL9 isshown in SEQ ID NO: 1. The nucleotide sequence of the gene encoding theV region of the H chain of the mouse anti-HM1.24 antibody contained inthe plasmid pUCHMVHR16 is shown in SEQ ID NO: 3.

Reference Example 3 Determination of CDR

The overall structures of the V regions of an L chain and an H chainhave a similarity with each other in which four framework portions arelinked by three hypervariable regions, i.e. complementarity determiningregions (CDR). The amino acid sequence of the framework is relativelywell conserved but variation in the amino acid sequence is extremelyhigh (Kabat, E. A., et al., “Sequences of Proteins of ImmunologicalInterest”, US Dept. Health and Human Services, 1983).

Based on these facts, the amino acid sequence of the variable region ofthe anti-HM1.24 antibody was fitted to the database of the amino acidsequences of antibodies to investigate homology, and the CDR region wasdetermined as shown in Table 1.

TABLE 1 Plasmid Sequence No. CDR(1) CDR(2) CDR(3) pUCHMVL9 5 to 7  24–3450–56 89–97  pUCHMVHR16 8 to 10 31–35 50–66 99–109

Reference Example 4 Confirmation of Expression of the Cloned cDNA(Construction of the Chimera Anti-HM1.24 Antibody)

1. Construction of an Expression Vector

In order to construct an expression vector that expresses a chimeraanti-HM1.24 antibody, cDNA clones pUCHMVL9 and pUCHMVHR16 encoding the Vregions of the L chain and the H chain of the mouse anti-HM1.24antibody, respectively, were modified by the PCR method, and thenintroduced into the HEF expression vector (International PatentPublication No. WO 92-19759).

The backward primer ONS-L722S (SEQ ID NO: 65) for the V region of an Lchain and the backward primer VHR16S (SEQ ID NO: 66) for the V region ofan H chain were designed so that they hybridize to the DNA encoding thestart of the leader sequence of the V region of each and they have theKozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196, 947–950,(1987)) and the recognition site for HindIII restriction enzyme. Theforward primer VL9A (SEQ ID NO: 67) for the V region of an L chain andthe forward primer VHR16A (SEQ ID NO: 68) for the V region of an H chainwere designed so that they hybridize to the DNA sequence encoding theend of the J region and they have a splice donor sequence and therecognition site for BamHI restriction enzyme.

One hundred μl of the PCR reaction mixture containing 10 mM Tris-HCl (pH8.3), 50 mM KCl, 0.1 mM dNTPs, 1.5 mM MgCl₂, 100 pmole each of eachprimer, 100 ng of template DNA (pUCHMVL9 or pUCHMVHR16), and 5 units ofAmpli Taq enzyme was covered with 50 μl of a mineral oil, and then afterthe initial denaturation at 94° C., heated at 94° C. for 1 minute, at55° C. for 1 minute and at 72° C. for 1 minute for 30 cycles and finallyincubated at 72° C. for 10 minutes.

The PCR product was purified by the low melting point agarose gel, anddigested with HindIII and BamHI, and then cloned to HEF-VL-gκ for the Vregion of the L chain and to HEF-VH-gγ1 for the V region of the H chain.After determination of the DNA sequence, the plasmids containing the DNAfragment that contains the correct DNA sequence were designated asHEF-1.24L-gκ and HEF-1.24H-gγl, respectively.

The regions encoding the respective variable region from the aboveplasmids HEF-1.24L-gκ and HEF-1.24H-gγl were digested with restrictionenzymes HindIII and BamHI to make restriction fragments, which wereinserted to the HindIII site and the BamHI sites of plasmid vector pUC19and they were designated as pUC19-1.24L-gκ and pUC19-1.24H-gγl,respectively.

Escherichia coli containing respective plasmids pUC19-1.24L-gκ andpUC19-1.24H-gγl were designated as Escherichia coli DH5 (pUC19-1.24L-gκ)and Escherichia coli DH5 (pUC19-1.24H-gγl), and were internationallydeposited on Aug. 29, 1996, with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under theaccession numbers FERM BP-5646 and FERM BP-5644, respectively, under theprovisions of the Budapest Treaty.

2. Transfection into COS-7 Cells

In order to observe the transient expression of the chimera anti-HM1.24antibody, the above expression vectors were tested in the COS-7 (ATCCCRL-1651) cells. HEF-1.24L-gκ and HEF-1.24H-gγl were cotransformed intoCOS-7 cells by electroporation using the Gene Pulser instrument(manufactured by BioRad). Each DNA (10 μg) was added to 0.8 ml aliquotsof 1×10⁷ cells/ml in PBS, and was subjected to pulses at 1500 V and acapacity of 25 μF.

After a recovery period of 10 minutes at room temperature, theelectroporated cells were added to 30 ml of the DHEM culture liquid(manufactured by GIBCO) containing 10% γ-globulin-free bovine fetalserum. After incubation of 72 hours in the CO₂ incubator BNA120D(manufactured by TABAI), the culture supernatant was collected, the celldebris was removed by centrifugation, and the supernatant was used forthe following experiment.

3. FCM Analysis

The antigen binding activity of the chimera anti-HM1.24 antibody wasinvestigated by FCM (flow cytometry) analysis using the KPMM2 cells.After 4.7×10⁵ KPMM2 cells (Japanese Unexamined Patent Publication(Kokai) No. 7(1995)-236475) were washed with PBS(−), 50 μl of theculture of COS-7 cells that produce the above-mentioned chimeraanti-HM1.24 antibody and 50 μl of FACS buffer (PBS(−) containing 2%bovine fetal serum and 0.1% sodium azide), or 5 μl of 500 μg/ml purifiedmouse anti-HM1.24 antibody and 95 μl of the FACS buffer were added, andincubated at the temperature of ice for one hour.

As a control, 50 μl of 2 μg/ml chimera SK2 (International PatentPublication No. WO 94-28159) and 50 μl of the FACS buffer, or 5 μl of500 μg/ml purified mouse IgG2aκ (UPC10) (manufactured by CAPPEL) insteadof purified mouse anti-HM1.24 antibody, and 95 μl of FACS buffer wereadded, and similarly incubated. After washing with the FACS buffer, 100μl of 25 μg/ml FITC-labeled goat anti-human antibody (GAH) (manufacturedby CAPPEL) or 10 μg/ml FITC labeled goat anti-mouse antibody (GAM)(manufactured by Becton Dickinson) were added, and incubated at atemperature of ice for 30 minutes. After washing with the FACS buffer,it was suspended in one ml of the FACS buffer, and fluorescenceintensity of each cell was measured by the FACScan (manufactured byBecton Dickinson).

As shown in FIG. 1, it was revealed that the chimera anti-HM1.24antibody bound to the KPMM2 cell because the peak of fluorescenceintensity shifted to the right in the chimera anti-HM1.24 antibody-addedcells as compared to the control similarly to the case where mouseanti-HM1.24 antibody was added. This confirmed that the cloned cDNAencodes the variable region of the mouse anti-HM1.24 antibody.

Reference Example 5 Establishment of the CHO Cell Line that StablyProduces a Chimera Anti-HM1.24 Antibody

1. Construction of an Expression Vector for the Chimera H Chain

By digesting the above plasmid HEF-1.24H-gγl with the restrictionenzymes PvuI and BamHI, an about 2.8 kbp fragment containing the EF1promoter and the DNA encoding the V region of the H chain of the mouseanti-HM1.24 antibody was purified using 1.5% low melting point agarosegel. Then, the above DNA fragment was inserted into an about 6 kbpfragment prepared by digesting the expression vector used for a human Hchain expression vector, DHFR-ΔE-Rvh-PM1f (see International PatentPublication No. WO 92/19759), containing the DHFR gene and the geneencoding the constant region of a human H chain with PvuI and BamHI toconstruct an expression vector, DHFR-ΔE-HEF-1.24-H-gγ1, for the H chainof the chimera anti-HM1.24 antibody.

2. Gene Introduction into CHO Cells

In order to establish a stable production system of the chimeraanti-HM1.24 antibody, the genes of the above-mentioned expressionvectors, HEF-1.24L-gκ and DHFR-ΔE-HEF-1.24H-gγl, that were linearized bydigestion with PvuI were simultaneously introduced into the CHO cellDXBII (donated from the Medical Research Council Collaboration Center)by the electroporation method under the condition similar to theabove-mentioned one (the above-mentioned transfection into the COS-7cells).

3. Gene Amplification by MTX

Among the gene-introduced CHO cells, only those CHO cells in which bothof the L chain and the H chain expression vectors have been introducedcan survive in the nucleoside-free α-MEM culture liquid (manufactured byGIBCO-BRL) to which 500 μg/ml G418 (manufactured by GIBCO-BRL) and 10%bovine fetal serum were added, and so they were selected. Subsequently,10 nM MTX (manufactured by Sigma) was added to the above culture liquid.Among the clones that propagated, those that produce the chimeraanti-HM1.24 antibody in large amounts were selected. As a result, clones#8 to #13 that exhibited a production efficiency of about 20 μg/ml ofthe chimera antibody were obtained and termed the chimera anti-HM1.24antibody-producing cell lines.

Reference Example 6 Construction of the Chimera Anti-HM1.24 Antibody

The chimera anti-HM1.24 antibody was constructed in the followingmethod. The above chimera anti-HM1.24 antibody-producing CHO cells weresubjected to continuous culture for 30 days using as the medium Iscove'sModified Dulbecco's Medium (manufactured by GIBCO-BRL) containing 5%γ-globulin-free newborn bovine serum (manufactured by GIBCO-BRL) by thehigh-density cell culture instrument Verax system 20 (manufactured byCELLEX BIOSCIENCE Inc.).

On day 13, 20, 23, 26, and 30 after starting the culture, the cultureliquid was recovered using a pressurized filter unit SARTOBRAN(manufactured by Sartorius), and then the chimera anti-HM1.24 antibodywas affinity-purified using a large-volume antibody collection systemAfi-Prep System (manufactured by Nippon Gaishi) and Super Protein Acolumn (bed volume: 100 ml, manufactured by Nippon Gaishi) using PBS asthe absorption/wash buffer and 0.1 M sodium citrate buffer (pH 3) as theelution buffer according to the attached instructions. The elutedfractions were adjusted to about pH 7.4 by immediately adding 1 MTris-HCl (pH 8.0). Antibody concentration was measured by absorbance at280 nm and calculated with 1 μg/ml as 1.35 OD.

Reference Example 7 Determination of Activity of the Chimera Anti-HM1.24Antibody

Chimera anti-HM1.24 antibody was evaluated by the following bindinginhibition activity.

1. Measurement of Binding Inhibition Activity

1-1. Construction of a Biotinylated Anti-HM1.24 Antibody

After the mouse anti-HM1.24 antibody was diluted with 0.1 M bicarbonatebuffer to 4 mg/ml, 4 μl of 50 mg/ml Biotin-N-hydroxy succinimide(manufactured by EY LABS Inc.) was added and reacted at room temperaturefor 3 hours. Thereafter, 1.5 ml of 0.2 M glycine solution was addedthereto, incubated at room temperature for 30 minutes to stop thereaction, and then the biotinylated IgG fractions were collected usingthe PD-10 column (manufactured by Pharmacia Biotech).

1-2. Measurement of Binding Inhibition Activity

The binding inhibition activity by the biotin-labeled mouse anti-HM1.24antibody was measured by the Cell-ELISA using the human amnioticmembrane cell line WISH cells (ATCC CCL 25). The Cell-ELISA plates wereprepared as follows. To a 96-well plate was added 4×10⁵ cells/mlprepared with PRMI 1640 medium supplemented with 10% fetal bovine serum,incubated overnight, and after washing twice with PBS(−), wereimmobilized with 0.1% glutaraldehyde (manufactured by Nacalai TesqueInc.).

After blocking, 50 μl of serial dilutions of the chimera anti-HM1.24antibody or the mouse anti-HM1.24 antibody obtained by affinitypurification was added to each well and simultaneously 50 μl of 2 μg/mlbiotin-labeled mouse anti-HM1.24 antibody was added, incubated at roomtemperature for two hours, and then the peroxidase-labeled streptavidin(manufactured by DAKO) was added. After incubating at room temperaturefor one hour and then washing, the substrate solution was added. Afterstopping the reaction by adding 50 μl of 6N sulfuric acid, absorbance at490 nm was measured using the MICROPLATE READER Model 3550 (manufacturedby Bio-Rad).

The result, as shown in FIG. 2, revealed that the chimera anti-HM1.24antibody has a similar binding inhibition activity with the mouseanti-HM1.24 antibody as the biotin-labeled mouse anti-HM1.24 antibody.This indicates that the chimera antibody had the same V region as themouse anti-HM1.24 antibody.

Reference Example 8 Measurement of the ADCC Activity of the ChimeraAnti-HM1.24 Antibody

ADCC (Antibody-dependent Cellular Cytotoxicity) activity was measuredaccording to the method as set forth in Current Protocols in Immunology,Chapter 7, Immunologic studies in humans, Editor, Johan E. Coligan etal., John Wiley & Sons, Inc., 1993.

1. Preparation of Effector Cells

Monocytes were separated from the peripheral blood or bone marrow ofhealthy humans and patients with multiple myeloma by the densitycentrifugation method. Thus, an equal amount of PBS(−) was added to theperipheral blood and the bone marrow of healthy humans and patients withmultiple myeloma, which was layered on Ficoll (manufactured byPharmacia)-Conrey (manufactured by Daiichi Pharmaceutical Co. Ltd.)(specific gravity, 1.077), and was centrifuged at 400 g for 30 minutes.The monocyte layer was collected, and washed twice with RPMI 1640(manufactured by Sigma) supplemented with 10% bovine fetal serum(manufactured by Witaker), and prepared at a cell density of 5×10⁶/mlwith the same culture liquid.

2. Preparation of Target Cells

The human myeloma cell line RPMI 8226 (ATCC CCL 155) was radiolabeled byincubating in the RPMI 1640 (manufactured by Sigma) supplemented with10% bovine fetal serum (manufactured by Witaker) together with 0.1 mCiof ⁵¹Cr-sodium chromate at 37° C. for 60 minutes. After radiolabeling,cells were washed three times with Hanks balanced salt solution (HBSS)and adjusted to a concentration of 2×10⁵/ml.

3. ADCC Assay

Into a 96-well U-bottomed plate (manufactured by Corning) were added 50μl of 2×10⁵ target cells/ml, 1 μg/ml of affinity-purified chimeraanti-HM1.24 antibody and mouse anti-HM1.24 antibody, or control humanIgG (manufactured by Serotec), and the plate was held at 4° C. for 15minutes.

Then, 100 μl of 5×10⁵ effector cells/ml was added thereto, and theresult was cultured in a CO₂ incubator for 4 hours, whereupon the ratio(E:T) of the effector cells (E) to the target cells (T) was set at 0:1,5:1, 20:1, or 50:1.

One hundred μl of the supernatant was taken and the radioactivityreleased into the culture supernatant was measured by a gamma counter(ARC361, manufactured by Aloka). For measurement of the maximumradioactivity, 1% NP-40 (manufactured by BRL) was used. Cytotoxicity (%)was calculated by (A−C)/(B−C)×100, wherein A is radioactivity (cpm)released in the presence of antibody, B is radioactivity (cpm) releasedby NP-40, and C is radioactivity (cpm) released by the culture liquidalone without antibody.

As shown in FIG. 3, when the chimera anti-HM1.24 antibody was added ascompared to the control IgG1, cytotoxicity increased with the increasein the E:T ratio, which indicated that this chimera anti-HM1.24 antibodyhas ADCC activity. Furthermore, since there was no cytotoxicity observedeven when the mouse anti-HM1.24 antibody was added, it was shown thatthe Fc portion of human antibody is required to obtain ADCC activitywhen the effector cell is a human-derived cell.

Reference Example 9 Construction of the Reshaped Human Anti-HM1.24Antibody

1. Designing of the V Region of the Reshaped Human Anti-HM1.24 Antibody

In order to construct the reshaped human antibody in which the CDR ofmouse monoclonal antibody has been transplanted to a human antibody, itis preferred that there is a high homology between the FR of the mouseantibody and the FR of the human antibody. Thus, the V regions of the Lchain and the H chain of the mouse anti-HM1.24 antibody were compared tothe V regions of all known antibodies whose structure has beenelucidated using the Protein Data Bank.

The V region of the L chain of the mouse anti-HM1.24 antibody is mostsimilar to the consensus sequence of the subgroup IV (HSGIV) of the Vregion of a human L chain with a homology of 66.4%. On the other hand,it has shown a homology of 56.9%, 55.8%, and 61.5% with HSGI, HSGII andHSG III, respectively.

When the V region of the L chain of the mouse anti-HM1.24 antibody iscompared to the V region of the L chain of known human antibodies, ithas shown a homology of 67.0% with the V region REI of a human L chain,one of the subgroups I of the V region of a human L chain. Thus, the FRof REI was used as the starting material for construction of the Vregion of the L chain of the reshaped human anti-HM1.24 antibody.

Version a of the L chain V region of the reshaped human anti-HM1.24antibody was designed. In this version, human FR was made identical withthe REI-based FR present in the reshaped human CAMPATH-1H antibody (seeRiechmann, L. et al., Nature 322, 21–25, (1988), the FR contained inversion a of the V region of the L chain of the reshaped human anti PM-1antibody described in International Patent Publication No. WO 92-19759),and the mouse CDR was made identical with the CDR in the V region of theL chain of the mouse anti-HM1.24 antibody.

The H chain V region of the mouse anti-HM1.24 antibody is most similarto the consensus sequence of HSGI of the V region of a human H chainwith a homology of 54.7%. On the other hand, it shows a homology of34.6% and 48.1% with HSGII and HSGIII, respectively. When the V regionof the H chain of the mouse anti-HM1.24 antibody is compared to the Vregion of the H chain of known human antibodies, FR1 to FR3 were mostsimilar to the V region of the H chain of the human antibody HG3, one ofsubgroup I of the V region of a human H chain (Rechavi, G. et al., Proc.Natl. Acad. Sci. USA, 80, 855–859), with a homology of 67.3%.

Therefore, the FR of the human antibody HG3 was used as the startingmaterial for construction of the V region of the H chain of the reshapedhuman anti-HM1.24 antibody. However, since the amino acid sequence ofthe FR4 of human HG3 has not been described, the amino acid sequence ofthe FR4 of the human antibody JH6 (Ravetch, J. V. et al., Cell, 27,583–591) that shows the highest homology with the FR4 of the H chain ofthe mouse anti-HM1.24 antibody was used. The FR4 of JH6 has the sameamino acid sequence as that of the FR4 of the H chain of the mouseanti-HM1.24 antibody except for one amino acid.

In the first version a of the V region of the H chain of the reshapedhuman anti-HM1.24 antibody, FR1 to FR3 were made identical with the FR1to FR3 of human HG3, and the CDR was made identical with the CDR of theV region of the H chain of the mouse anti-HM1.24 antibody, except thatthe amino acids at position 30 in the human FR1 and position 71 in thehuman FR3 were made identical with the amino acids in the mouseanti-HM1.24 antibody.

2. Construction of the V Region of the L Chain of the Reshaped HumanAnti-HM1.24 Antibody

The L chain of the reshaped human anti-HM1.24 antibody was constructedby the CDR grafting in the PCR method. The method is shown in FIG. 4.Eight PCR primers were used for construction of the reshaped humananti-HM1.24 antibody (version a) having the FR derived from the humanantibody REI. The external primers A (SEQ ID NO: 69) and H (SEQ ID NO:70) were designed to hybridize with the DNA sequence of the expressionvector HEF-VL-gκ.

The CDR grafting primers L1S (SEQ ID NO: 71), L2S (SEQ ID NO: 72), andL3S (SEQ ID NO: 73) have the sense DNA sequence. The CDR graftingprimers L1A (SEQ ID NO: 74), L2A (SEQ ID NO: 75), and L3A (SEQ ID NO:76) have the antisense DNA sequence, each having a complementary DNAsequence (20 to 23 bp) to the DNA sequence at the 5′-end of the primersL1S, L2S, and L3S, respectively.

In the first stage of PCR, the four reactions A-L1A, L1S-L2A, L2S-L3A,and L3S-H were conducted to purify each PCR product. The four PCRproducts from the first PCR were allowed to assemble with one another bytheir own complementarity (see International Patent Publication No. WO92-19759). Then, external primers A and H were added to amplify thefull-length DNA encoding the V region of the L chain of the reshapedhuman anti-HM1.24 antibody (the second PCR). In the above-mentioned PCR,the plasmid HEF-RVL-M21a (see International Patent Publication No. WO95-14041) encoding the version a of the V region of the L chain of thereshaped human ONS-M21 antibody based on the human antibody REI-derivedFR was employed as a template.

In the first stage of PCR, the PCR mixture containing 10 mM Tris-HCl (pH8.3), 50 mM KCl, 0.1 mM dNTPs, 1.5 mM MgCl2, 100 ng of template DNA, 100pmole of each primer, and 5 u of Ampli Taq was used. Each PCR tube wascovered with 50 μl of a mineral oil. Then after it was first denaturedby heating at 94° C., it was subjected to a reaction cycle of 94° C. for1 minute, 55° C. for 1 minute and 72° C. for 1 minute, and then wasincubated at 72° C. for 10 minutes.

PCR products A-L1A (215 bp), L1S-L2A(98 bp), L2S-L3A (140 bp), and L3S-H(151 bp) were purified using 1.5% low melting point agarose gel and wereassembled in the second PCR. In the second PCR, 98 μl of PCR mixturecontaining 1 μg each of the first stage PCR products and 5 u of AmpleTaq was incubated for 2 cycles of 94° C. for 2 minutes, 55° C. for 2minutes, and 72° C. for 2 minutes, and then 100 pmole each of theexternal primers (A and H) was added. The PCR tube was coated with 50 μlof a mineral oil and 30 cycles of PCR were conducted under the samecondition as above.

A 516 bp DNA fragment resulting from the second PCR was purified using1.5% low melting point agarose gel, digested with BamHI and HindIII, andthe DNA fragments thus obtained were cloned into the HEF expressionvector HEF-VL-gκ. After determining the DNA sequence, the DNA fragmenthaving the correct amino acid sequence of the V region of the L chain ofthe reshaped human anti-HM1.24 antibody was designated as plasmidHEF-RVLa-AHM-gκ. The amino acid sequence and the nucleotide sequence ofthe V region of L chain contained in this plasmid HEF-RVLa-AHM-gκ areshown in SEQ ID NO: 11.

The version b of the V region of the L chain of the reshaped humananti-HM1.24 antibody was constructed by mutagenesis using PCR. Mutagenprimers FTY-1 (SEQ ID NO: 77) and FTY-2 (SEQ ID NO: 78) were so designedas to mutate phenylalanine at position 71 to tyrosine.

After the above primers were amplified using the plasmid HEF-RVLa-AHM-gκas a template, the final product was purified by digesting with BamHIand HindIII. The DNA fragments obtained were cloned into the HEFexpression vector HEF-VL-gκ to obtain plasmid HEF-RVLb-AHM-gκ. The aminoacid sequence and the base sequence of the V region of the L chaincontained in this plasmid HEF-RVLb-AHM-gκ are shown in SEQ ID NO: 13.

3. Construction of the H Chain V Region of the Reshaped HumanAnti-HM1.24 Antibody

3-1. Construction of Versions a to e of the H Chain V Region of theReshaped Human Anti-HM1.24 Antibody

DNA encoding the V region of the H chain of the reshaped humananti-HM1.24 antibody was designed as follows. By linking the DNAsequence encoding the FR1 to 3 of the human antibody HG3 and the FR4 ofthe human antibody JH6 to the DNA sequence encoding the CDR of the Vregion of the H chain of the mouse anti-HM1.24 antibody, the full lengthDNA encoding the V region of the H chain of the reshaped humananti-HM1.24 antibody was designed.

Then, to the 5′-end and the 3′-end of this DNA sequence the HindIIIrecognition site/KOZAK consensus sequence and BamHI recognitionsite/splice donor sequence, respectively, were attached so as to enableinsertion of the HEF expression vector.

The DNA sequence thus designed was divided into four oligonucleotides.Subsequently, oligonucleotides which potentially hinder assembly ofthese oligonucleotides were subjected to computer analysis for thesecondary structure. The sequences of the four oligonucleotides RVH1 toRVH4 are shown in SEQ ID NO: 79 to 82. These oligonucleotides have alength of 119 to 144 bases and have the 25 to 26 bp overlapping region.Among the oligonucleotides, RVH2 (SEQ ID NO: 80) and RVH4 (SEQ ID NO:82) have the sense DNA sequence, and RVH1 (SEQ ID NO: 79) and RVH3 (SEQID NO: 81) have the antisense DNA sequence. The method for assemblingthese four oligonucleotides by the PCR method is shown in the figure(see FIG. 5).

The PCR mixture (98 μl) containing 100 ng each of the fouroligonucleotides and 5 u of Ampli Taq was first denatured by heating at94° C. for 2 minutes, and was subjected to two cycles of incubationcomprising 94° C. for 2 minutes, 55° C. for 2 minutes and 72° C. for 2minutes. After 100 pmole each of RHP1 (SEQ ID NO: 83) and RHP2 (SEQ IDNO: 84) were added as the external primer, the PCR tube was coated with50 μl of a mineral oil. Then it was first denatured by heating at 94° C.for 1 minute, and then was subjected to 38 cycles of 94° C. for 1minute, 55° C. for 1 minute and 72° C. for 1 minute, and then wasincubated at 72° C. for 10 minutes.

The 438 bp DNA fragment was purified using 1.5% low melting pointagarose gel, digested with HindIII and BamHI, and then cloned into theHEF expression vector HEF-VH-gγ1. After determination of the basesequence, the plasmid that contains the DNA fragment encoding the aminoacid sequence of the correct V region of the H chain was designated asHEF-RVHa-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHa-AHM-gγ1 areshown in SEQ ID NO: 11.

Each of versions b, c, d, and e of the V region of the H chain of thereshaped human anti-HM1.24 antibody was constructed as follows.

Using as the mutagen primer BS (SEQ ID NO: 85) and BA (SEQ ID NO: 86)designed to mutate arginine at position 66 to lysine and, as a templateDNA, the plasmid HEF-RVHa-AHM-gγ1 by the PCR method, version b wasamplified to obtain plasmid HEF-RVHb-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHb-AHM-gγ1 are shown in SEQ ID NO: 17.

Using as the mutagen primer CS (SEQ ID NO: 87) and CA (SEQ ID NO: 88)designed to mutate threonine at position 73 to lysine and, as a templateDNA, the plasmid HEF-RVHa-AHM-gγ1 by the PCR method, version c wasamplified to obtain plasmid HEF-RVHc-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHc-AHM-gγ1 are shown in SEQ ID NO: 19.

Using as the mutagen primer DS (SEQ ID NO: 89) and DA (SEQ ID NO: 90)designed to mutate arginine at position 66 to lysine and threonine atposition 73 to lysine and as a template DNA the plasmid HEF-RVHa-AHM-gγ1by the PCR method, version d was amplified to obtain plasmidHEF-RVHd-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHd-AHM-gγ1 areshown in SEQ ID NO: 21.

Using as the mutagen primer ES (SEQ ID NO: 91) and EA (SEQ ID NO: 92)designed to mutate valine at position 67 to alanine and methionine atposition 69 to leucine and as a template DNA the plasmidHEF-RVHa-AHM-gγ1, version e was amplified to obtain plasmidHEF-RVHe-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHe-AHM-gγ1 areshown in SEQ ID NO: 23.

3-2. Construction of the H Chain Hybrid V Region

Two H chain hybrid V regions were constructed. One is a mouse-humanhybrid anti-HM1.24 antibody in which the amino acid sequences of FR1 andFR2 are derived from the mouse anti-HM1.24 antibody and those of FR3 andFR4 are from version a of the V region of the H chain of the reshapedhuman anti-HM1.24 antibody, and the other is human-mouse hybridanti-HM1.24 antibody in which the amino acid sequences of FR1 and FR2are derived from version a of the V region of the H chain of thereshaped human anti-HM1.24 antibody and those of FR3 and FR4 are fromthe mouse anti-HM1.24 antibody. The amino acid sequences of the CDRregions are all derived from mouse anti-HM1.24 antibody.

Two H chain hybrid V regions were constructed by the PCR method. Themethod is schematically shown in FIGS. 6 and 7. For the construction oftwo H chain hybrid V regions, four primers were used. The externalprimers a (SEQ ID NO: 93) and h (SEQ ID NO: 94) were designed tohybridize with the DNA sequence of the HEF expression vector HEF-VH-gγ1.The H chain hybrid construction primer HYS (SEQ ID NO: 95) was designedto have the sense DNA sequence and the H chain hybrid primer HYA (SEQ IDNO: 96) to have the antisense DNA sequence so that the DNA sequence arecomplementary to each other.

For the construction of the H chain hybrid V region in which the aminoacid sequences of FR1 and FR2 are derived from the mouse anti-HM1.24antibody and those of FR3 and FR4 are from version a of the V region ofthe H chain of the reshaped human anti-HM1.24 antibody, PCR using theplasmid HEF-1.24H-gγl as a template, the external primer a, and the Hchain hybrid primer HYA, and PCR using the plasmid HEF-RVLa-AHM-gγ1 as atemplate, the H chain hybrid primer HYS (SEQ ID NO: 95), and theexternal primer h (SEQ ID NO: 94) were carried out in the first stage ofPCR to purify each PCR product. The two PCR products from the first PCRwere allowed to assemble by their own complementarity (see InternationalPatent Publication No. WO 92-19759).

Then, by adding the external primers a (SEQ ID NO: 93) and h (SEQ ID NO:94) a full-length DNA encoding the H chain hybrid V region in which theamino acid sequences of FR1 and FR2 are derived from the mouseanti-HM1.24 antibody and those of FR3 and FR4 are from version a of theV region of the H chain of the reshaped human anti-HM1.24 antibody wasamplified in the second PCR stage.

For the construction of the H chain hybrid V region in which the aminoacid sequences of FR1 and FR2 are derived from version a of the V regionof the H chain of the reshaped human anti-HM1.24 antibody and those ofFR3 and FR4 are from the mouse anti-HM1.24 antibody, PCR using theplasmid HEF-RVHa-AHM-gγ1 as a template, the external primer a, and the Hchain hybrid primer HYA, and PCR using the plasmid HEF-1.24H-gγl as atemplate, the H chain hybrid primer HYS, and the external primer h werecarried out in the first stage of PCR to purify each PCR product. Thetwo PCR purified products from the first PCR were allowed to assemble bytheir own complementarity (see International Patent Publication No. WO92-19759).

Then, by adding the external primers a and h, a full-length DNA encodingthe H chain hybrid V region in which the amino acid sequences of FR1 andFR2 are derived from version a of the V region of the H chain of thereshaped human anti-HM1.24 antibody and those of FR3 and FR4 are fromthe mouse anti-HM1.24 antibody was amplified in the second PCR stage.

The methods of the first PCR, purification of PCR products, assembling,the second PCR, and cloning into the HEF expression vector HEF-VH-gγ1were carried out according to the methods shown in “Example 9.Construction of the V region of the L chain of the reshaped humananti-HM1.24 antibody”.

After sequencing the DNA sequence, the plasmid that contains the DNAfragment encoding the correct amino acid sequence of the H chain hybridV region in which the amino acid sequences of FR1 and FR2 are derivedfrom the mouse anti-HM1.24 antibody and those of FR3 and FR4 are fromversion a of the V region of the H chain of the reshaped humananti-HM1.24 antibody was termed HEF-MH-RVH-AHM-gγ1. The amino acidsequence and the base sequence of the V region of the H chain containedin this plasmid HEF-MH-RVH-AHM-gγ1 are shown in SEQ ID NO: 97. Also, theplasmid that contains the DNA fragment encoding the correct amino acidsequence of the H chain hybrid V region in which the amino acidsequences of FR1 and FR2 are derived from version a of the V region ofthe H chain of the reshaped human anti-HM1.24 antibody and those of FR3and FR4 are from the mouse anti-HM1.24 antibody was termedHEF-HM-RVH-AHM-gγ1. The amino acid sequence and the base sequence of theV region of the H chain contained in this plasmid HEF-HM-RVH-AHM-gγ1 areshown in SEQ ID NO: 99.

3-3. Construction of Versions f to r of the V Region of the H Chain ofthe Reshaped Human Anti-HM1.24 Antibody

Each of versions f, g, h, i, j, k, l, m, n, o, p, q, and r of the Vregion of the H chain of the reshaped human anti-HM1.24 antibody wereconstructed as follows.

Using as the mutagen primer FS (SEQ ID NO: 102) and FA (SEQ ID NO: 103)designed to mutate threonine at position 75 to serine and valine atposition 78 to alanine and as a template DNA the plasmidHEF-RVHe-AHM-gγ1 by the PCR method, version f was amplified to obtainplasmid HEF-RVHf-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHf-AHM-gγ1 are shown in SEQ ID NO: 25.

Using as the mutagen primer GS (SEQ ID NO: 104) and GA (SEQ ID NO: 105)designed to mutate alanine at position 40 to arginine and, as a templateDNA, the plasmid HEF-RVHa-AHM-gγ1, version g was amplified to obtainplasmid HEF-RVHg-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHg-AHM-gγ1 are shown in SEQ ID NO: 27.

Using as the mutagen primer FS (SEQ ID NO: 102) and FA (SEQ ID NO: 103)and, as a template DNA, the plasmid HEF-RVHb-AHM-gγ1, version h wasamplified to obtain plasmid HEF-RVHh-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHh-AHM-gγ1 are shown in SEQ ID NO: 29.

Using as the mutagen primer IS (SEQ ID NO: 106) and IA (SEQ ID NO: 107)designed to mutate arginine at position 83 to alanine and serine atposition 84 to phenylalanine and, as a template DNA, the plasmidHEF-RVHh-AHM-gγ1, version i was amplified to obtain plasmidHEF-RVHi-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHi-AHM-gγ1 areshown in SEQ ID NO: 31.

Using as the mutagen primer JS (SEQ ID NO: 108) and JA (SEQ ID NO: 109)designed to mutate arginine at position 66 to lysine and, as a templateDNA, the plasmid HEF-RVHf-AHM-gγ1, version j was amplified to obtainplasmid HEF-RVHj-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHj-AHM-gγ1 are shown in SEQ ID NO: 33.

Using as the mutagen primer KS (SEQ ID NO: 110) and KA (SEQ ID NO: 111)designed to mutate glutamic acid at position 81 to glutamine and, as atemplate DNA, the plasmid HEF-RVHh-AHM-gγ1, version k was amplified toobtain plasmid HEF-RVHk-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHk-AHM-gγ1 are shown in SEQ ID NO: 35.

Using as the mutagen primer LS (SEQ ID NO: 112) and LA (SEQ ID NO: 113)designed to mutate glutamic acid at position 81 to glutamine and serineat position 82B to isoleucine and, as a template DNA, the plasmidHEF-RVHh-AHM-gγ1, version 1 was amplified to obtain plasmidHEF-RVHl-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHl-AHM-gγ1 areshown in SEQ ID NO: 37.

Using as the mutagen primer MS (SEQ ID NO: 114) and MA (SEQ ID NO: 115)designed to mutate glutamic acid at position 81 to glutamine, serine atposition 82b to isoleucine, and threonine at position 87 to serine and,as a template DNA, the plasmid HEF-RVHh-AHM-gγ1, version m was amplifiedto obtain plasmid HEF-RVHm-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHm-AHM-gγ1 are shown in SEQ ID NO: 39.

Using as the mutagen primer NS (SEQ ID NO: 116) and NA (SEQ ID NO: 117)designed to mutate serine at position 82B to isoleucine and, as atemplate DNA, the plasmid HEF-RVHh-AHM-gγ1, version n was amplified toobtain plasmid HEF-RVHn-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHn-AHM-gγ1 are shown in SEQ ID NO: 41.

Using as the mutagen primer OS (SEQ ID NO: 118) and OA (SEQ ID NO: 119)designed to mutate threonine at position 87 to serine and, as a templateDNA, the plasmid HEF-RVHh-AHM-gγ1, version o was amplified to obtainplasmid HEF-RVHo-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHo-AHM-gγ1 are shown in SEQ ID NO: 43.

Using as the mutagen primer PS (SEQ ID NO: 120) and PA (SEQ ID NO: 121)designed to mutate valine at position 78 to alanine and, as a templateDNA, the plasmid HEF-RVHa-AHM-gγ1, version p was amplified by the PCRmethod to obtain plasmid HEF-RVHp-AHM-gγ1. The amino acid sequence andthe base sequence of the V region of the H chain contained in thisplasmid HEF-RVHp-AHM-gγ1 are shown in SEQ ID NO: 45.

Using as the mutagen primer QS (SEQ ID NO: 122) and QA (SEQ ID NO: 123)designed to mutate threonine at position 75 to serine and, as a templateDNA, the plasmid HEF-RVHa-AHM-gγ1, version q was amplified by the PCRmethod to obtain plasmid HEF-RVHq-AHM-gγ1. The amino acid sequence andthe base sequence of the V region of the H chain contained in thisplasmid HEF-RVHq-AHM-gγ1 are shown in SEQ ID NO: 47.

Using as the mutagen primer CS (SEQ ID NO: 87) and CA (SEQ ID NO: 88)and, as a template DNA, the plasmid HEF-RVHp-AHM-gγ1, version r wasamplified by the PCR method to obtain plasmid HEF-RVHr-AHM-gγ1. Theamino acid sequence and the base sequence of the V region of the H chaincontained in this plasmid HEF-RVHr-AHM-gγ1 are shown in SEQ ID NO: 49.

The regions encoding the variable region of each of the above-mentionedplasmids HEF-RVLa-AHM-gκ and HEF-RVHr-AHM-gγ1 were digested to makerestriction fragments with restriction enzymes HindIII and BamHI. Theywere inserted into the HindIII and BamHI sites of plasmid vector pUC19.Each plasmid was termed pUC19-RVLa-AHM-gκ and pUC19-RVHr-AHM-gγ1.

The Escherichia coli that contain each of the plasmids pUC19-RVLa-AHM-gκand pUC19-RVHr-AHM-gγ1 was termed Escherichia coli DH5α(pUC19-RVLa-AHM-gκ) and Escherichia coli DH5α (pUC19-RVHr-AHM-gγ1),respectively, and have been internationally deposited on Aug. 29, 1996,with the National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukubacity, Ibalaki prefecture, Japan) under the accession numbers FERMBP-5645 and FERM BP-5643, respectively, under the provisions of theBudapest Treaty.

4. Construction of the Reshaped Human Anti-HM1.24 Antibody, the ChimeraAnti-HM1.24 Antibody, and the H Chain Hybrid Antibody

In order to evaluate each chain of the reshaped human anti-HM1.24antibody, the reshaped human anti-HM1.24 antibody and the chimeraanti-HM1.24 antibody as a positive control antibody were allowed toexpress. In constructing each of version b and after of the V region ofthe H chain of the reshaped human anti-HM1.24 antibody, the H chainhybrid antibody was allowed to express in order to investigate whichamino acid sequence in the FR should be substituted. Furthermore, it wasexpressed in combination with the chimera H chain in order to evaluateversion a of L chain of the reshaped human anti-HM1.24 antibody.

4-1. Expression of the Reshaped Human Anti-HM1.24 Antibody

Ten μg each of the expression vector (HEF-RVHa-AHM-gγ1 toHEF-RVHr-AHM-gγ1) for the H chain of the reshaped human anti-HM1.24antibody and the expression vector (HEF-RVLa-AHM-gκ or HEF-RVLb-AHM-gκ)for the L chain of the reshaped human anti-HM1.24 antibody werecotransformed into COS-7 cells by electroporation using the Gene Pulserinstrument (manufactured by BioRad). Each DNA (10 μg) was added to 0.8ml aliquots of 1×10⁷ cells/ml in PBS, and was subjected to pulses at1500 V and a capacity of 25 μF.

After the recovery period of 10 minutes at room temperature, theelectroporated cells were added to 30 ml of DHEM culture liquid(manufactured by GIBCO) containing 10% γ-globulin-free bovine fetalserum. After incubation of 72 hours in the CO₂ incubator BNA120D(manufactured by TABAI) under the condition of 37° C. and 5% CO₂, theculture supernatant was collected, the cell debris was removed bycentrifugation at 1000 rpm for 5 minutes in a centrifuge 15PR-22(manufactured by HITACHI) equipped with a centrifuge rotor 03(manufactured by HITACHI), and a microconcentrator (Centricon 100,manufactured by Amicon) was ultrafiltrated using a centrifuge J2-21(manufactured by BECKMAN) equipped with a centrifuge rotor JA-20.1(manufactured by BECKMAN) at a condition of 2000 rpm, and was used forCell-ELISA.

4-2. Expression of the Chimera Anti-HM1.24 Antibody

Using ten μg each of the expression vector

HEF-1.24H-gγl for the H chain of the chimera human anti-HM1.24 antibodyand the expression vector HEF-1.24L-gκ for the L chain of the chimerahuman anti-HM1.24 antibody, the chimera anti-HM1.24 antibody to be usedfor Cell-ELISA was prepared according to the above-mentioned method forexpression of the reshaped human anti-HM1.24 antibody.

4-3. Expression of the Anti-HM1.24 Antibody Comprising Version a of theHumanized L Chain and the Chimera H Chain

Using ten μg each of the expression vector HEF-1.24H-gγl for the H chainof the chimera human anti-HM1.24 antibody and the expression vectorHEF-RVLa-AHM-Gκ for version a of the L chain of the reshaped humananti-HM1.24 antibody, the anti-HM1.24 antibody comprising version a ofthe humanized L chain and the chimera H chain to be used for Cell-ELISAwas prepared according to the above-mentioned method for expression ofthe reshaped human anti-HM1.24 antibody.

4-4. Expression of the H Chain Hybrid Antibody

Using ten μg each of the expression vector (HEF-MH-RVH-AHM-gγ1 orHEF-HM-RVH-AHM-gγ1) for the V region of the H chain hybrid and theexpression vector HEF-RVLa-AHM-gκ for the L chain of the reshaped humananti-HM1.24 antibody, the H chain hybrid antibody to be used forCell-ELISA was prepared according to the above-mentioned method forexpression of the reshaped human anti-HM1.24 antibody.

4-5. Measurement of Antibody Concentration

Concentration of the antibody obtained was measured by ELISA. Each wellof a 96-well ELISA plate (Maxisorp, manufactured by NUNC) wasimmobilized by adding 100 μl of goat anti-human IgG antibody(manufactured by BIO SOURCE) prepared to a concentration of 1 μg/ml withthe coating buffer (0.1 M NaHCO₃, 0.02% NaN₃, pH 9.6) and incubating atroom temperature for one hour. After blocking with 100 μl of thedilution buffer (50 mM Tris-HCl, 1 mM MgCl₂, 0.15 M NaCl, 0.05% Tween20, 0.02% NaN₃, 1% bovine serum albumin (BSA), pH 8.1), 100 μl each ofserial dilutions of the reshaped human anti-HM1.24 antibody, chimeraanti-HM1.24 antibody, and the H chain hybrid antibody that wereconcentrated by ultrafiltration were added to each well and incubated atroom temperature for one hour. Then, after washing, 100 μl of alkalinephosphatase-labeled goat anti-human IgG antibody (manufactured by DAKO)was added.

After incubating at room temperature for one hour and washing, 100 μl of1 μg/ml substrate solution (Sigma104, p-nitrophenyl phosphate,manufactured by SIGMA) dissolved in the substrate buffer (50 mM NaHCO₃,10 mM MgCl₂, pH 9.8) was added, and then the absorbance at 405 nm wasmeasured using the MICROPLATE READER Model 3550 (manufactured by BioRad). As the standard for the measurement of concentration, human IgG1κ(manufactured by The Binding Site) was used.

5. Establishment of the CHO Cell Line that Stably Produces the HumanAnti-HM1.24 Antibody

5-1. Construction of the Expression Vector for the H Chain of theReshaped Human Anti-HM1.24 Antibody

By digesting plasmid HEF-RVHr-AHM-gγ1 with the restriction enzymes PvuIand BamHI, an about 2.8 kbp fragment containing the DNA encoding the EF1promoter and the V region of the H chain of the reshaped humananti-HM1.24 antibody was purified using 1.5% low melting point agarosegel. Then, the above DNA fragment was inserted into an about 6 kbpfragment that was prepared by digesting the expression vector used for ahuman H chain expression vector, DHFR-ΔE-RVh-PM1f (International PatentPublication No. WO 92-19759), containing the DHFR gene and the geneencoding the constant region of a human H chain with PvuI and BamHI toconstruct an expression vector, DHFR-ΔE-HEF-RVHr-AHM-gγ1, for the Hchain of the reshaped anti-HM1.24 antibody.

5-2. Gene Introduction into CHO Cells

In order to establish a stable production system of the reshapedanti-HM1.24 antibody, the genes of the above-mentioned expressionvectors, DHFR-ΔE-RVHr-AHM-gγl and HEF-RVLa-AHM-gκ, that were linearizedby digestion with PvuI were simultaneously introduced into the CHO cellDXB-11 by the electroporation method under the condition similar to theabove-mentioned one (transfection into the above-mentioned COS-7 cells).

5-3. Gene Amplification by MTX

Among the gene-introduced CHO cells, only those CHO cells in which bothof L chain and H chain expression vectors have been introduced cansurvive in the nucleoside-free α-MEM culture liquid (manufactured byGIBCO-BRL) to which 500 μg/ml G418 (manufactured by GIBCO-BRL) and 10%bovine fetal serum were added, and so they were selected. Subsequently,10 nM MTX (manufactured by Sigma) was added to the above culture liquid.Among the clones that propagated, those that produce the reshapedanti-HM1.24 antibody in large amounts were selected. As a result, clone#1 that exhibits a production efficiency of about 3 μg/ml of thereshaped anti-HM1.24 antibody was obtained and termed the reshapedanti-HM1.24 antibody-producing cell line.

5-4. Construction of the Reshaped Human Anti-HM1.24 Antibody

The reshaped anti-HM1.24 antibody was constructed in the followingmethod. The above CHO cells that produce the reshaped anti-HM1.24antibody were cultured for 10 days using as the medium thenucleoside-free α-MEM culture liquid (manufactured by GIBCO-BRL) towhich 500 μg/ml G418 (manufactured by GIBCO-BRL) containing 10%γ-globulin-free bovine fetal serum (manufactured by GIBCO-BRL) wereadded using the CO₂ incubator BNAS120D (manufactured by TABAI) under thecondition of 37° C. and 5% CO₂. On day 8 and 10 after starting theculture the culture liquid was recovered, the cell debris was removed bycentrifuging for 10 minutes at 2000 rpm using the centrifuge RL-500SP(manufactured by Tomy Seiko) equipped with the TS-9 rotor, and thenfilter-sterilized using a bottle top filter (manufactured by FALCON)having a membrane with pores of 0.45 μm in diameter.

After an equal amount of PBS(−) was added to the culture liquid of theCHO cells that produce the reshaped human anti-HM1.24 antibody, then thereshaped anti-HM1.24 antibody was affinity-purified using the high-speedantibody purification system ConSep LC100 (manufactured by MILLIPORE)and Hyper D Protein A column (manufactured by Nippon Gaishi) usingPBS(−) as the absorption/wash buffer and 0.1 M sodium citrate buffer (pH3) as the elution buffer according to the attached instructions. Theeluted fractions were adjusted to about pH 7.4 by immediately adding 1 MTris-HCl (pH 8.0) and then using the centrifuging ultrafiltrationconcentrator Centriprep 10 (manufactured by MILLIPORE), concentrationand substitution to PBS(−) was carried out and filter-sterilized using amembrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore sizeof 0.22 μm to obtain the purified reshaped human anti-HM1.24 antibody.Antibody concentration was measured by absorbance at 280 nm andcalculated with 1 μg/ml as 1.35 OD.

Reference Example 11 Determination of Activity of the ReshapedAnti-HM1.24 Antibody

The reshaped anti-HM1.24 antibody was evaluated for the followingantigen binding activity and binding inhibition activity.

1. The Method of Measurement of Antigen Binding Activity and BindingInhibition Activity

1-1. Measurement of Antigen Binding Activity

Antigen binding activity was measured by the Cell-ELISA using WICHcells. Cell-ELISA plates were prepared as described in the above Example7.1-2.

After blocking, 100 μl of serial dilutions of the reshaped humananti-HM1.24 antibody that was obtained from the concentrate of theculture supernatant of COS-7 cells or purified from the culturesupernatant of CHO cells was added to each well. After it was incubatedfor 2 hours at room temperature and washed, peroxidase-labeled rabbitanti-human IgG antibody (manufactured by DAKO) was added. Afterincubating for 2 hours at room temperature and washing, the substratesolution was added and incubated. Then the reaction was stopped byadding 50 μl of 6N sulfuric acid, and absorbance at 490 nm was measuredusing the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

1-2. Measurement of Binding Inhibition Activity

The binding inhibition activity by the biotin-labeled mouse anti-HM1.24antibody was measured by the Cell-ELISA using WISH cells. Cell-ELISAplates were prepared as described above. After blocking, 50 μl of serialdilutions of the reshaped human anti-HM1.24 antibody that was obtainedfrom the concentrate of the culture supernatant of COS-7 cells orpurified from the culture supernatant of CHO cells was added to eachwell, and 50 μl of 2 μg/ml biotin-labeled mouse anti-HM1.24 antibody wasadded simultaneously. After incubating at room temperature for two hoursand washing, peroxidase-labeled streptavidin (manufactured by DAKO) wasadded. After incubating at room temperature for one hour and thenwashing, the substrate solution was added and incubated. Then thereaction was stopped by adding 50 μl of 6N sulfuric acid, and absorbanceat 490 nm was measured using the MICROPLATE READER Model 3550(manufactured by Bio-Rad).

2. Evaluation of the Reshaped Human Anti-HM1.24 Antibody

2-1. L Chain

Version a of the L chain of the reshaped human anti-HM1.24 antibody wasevaluated as mentioned above for measurement of antigen bindingactivity. As shown in FIG. 8, when version a of the L chain is expressedin combination with the chimera H chain it has shown a similar level ofantigen binding activity. However, in consideration of further increasein activity and of compatibility with the H chain, version b of the Lchain was constructed. Versions a and b of the L chain were evaluatedtogether for antigen binding activity and of binding inhibition activitywhen combined with versions a, b, f, or h of the H chain. As shown inFIGS. 9, 10, 11, and 12, version a of the L chain had a higher activitythan version b in both activities in all versions a, b, f, and h of theH chain. Therefore, version a of the L chain of the reshaped humananti-HM1.24 antibody was used for the following experiment.

2-2. H Chain Versions a to e

Versions a to e of the H chain of the reshaped human anti-HM1.24antibody were evaluated in combination with the version a of the L chainas mentioned above for measurement of antigen binding activity and forbinding inhibition activity. The result, as shown in FIGS. 11, 13, 14,and 15, indicated that all versions were weaker in both activities ascompared to the chimera anti-HM1.24 antibody, suggesting that furtheramino acid substitution is required.

2-3. The H Chain Hybrid Antibody

The H chain hybrid antibody was evaluated as mentioned above formeasurement of antigen binding activity. The result, as shown in FIG.16, indicated that the human-mouse hybrid anti-HM1.24 antibody has showna similar activity to that of the chimera anti-HM1.24 antibody forantigen binding activity, whereas the mouse-human hybrid anti-HM1.24antibody had a weaker activity than the chimera anti-HM1.24 antibody.This indicated that, in order to construct the reshaped humananti-HM1.24 antibody having the antigen binding activity similar to thatof the chimera anti-HM1.24 antibody, it is necessary to convert aminoacids included in FR3 or FR4 among those contained the V region of the Hchain.

2-4. Versions f to r of the H Chain

Version f of the H chain of the reshaped human anti-HM1.24 antibody wasevaluated as mentioned above for measurement of antigen bindingactivity. The result, as shown in FIG. 17, indicated that its antigenbinding activity is decreased as compared to the chimera anti-HM1.24antibody, but is increased as compared to the above versions a to c,suggesting that any of the four amino acids at positions 67, 69, 75, and78 that were newly converted in this version is responsible for theactivity of the reshaped human antibody.

Version g of the H chain of the reshaped human anti-HM1.24 antibody wasevaluated as mentioned above for measurement of antigen bindingactivity. The result, as shown in FIGS. 18 and 19, indicated that thisversion has exhibited a similar level of activity to that of the aboveversion a at most, revealing that, as shown for the above H chainhuman-mouse hybrid antibody, the amino acid at position 40 that wasconverted in this version is not responsible for the increase in theactivity of the reshaped human antibody.

Versions h to j of the H chain of the reshaped human anti-HM1.24antibody were evaluated as mentioned above for measurement of antigenbinding activity and of binding inhibition activity. The result, asshown in FIGS. 20, 21, 22, and 23, indicated that all versions wereweaker for both activities as compared to the chimera anti-HM1.24antibody and were similar to the above-mentioned f, suggesting that theamino acids at positions 67 and 69 among the four amino acids that werenewly converted in version f are not responsible for the increase in theactivity of the reshaped human antibody.

Versions k to p of the H chain of the reshaped human anti-HM1.24antibody were evaluated as mentioned above for measurement of antigenbinding activity and of binding inhibition activity. The result, asshown in FIGS. 24, 25, 26, and 27, indicated that all versions wereweaker for both activities as compared to the chimera anti-HM1.24antibody and were similar to the above-mentioned h, suggesting that theamino acids at position 80 and after that were newly converted in thesesix versions are not responsible for the increase in the activity of thereshaped human antibody.

Version q of the H chain of the reshaped human anti-HM1.24 antibody wasevaluated as mentioned above for measurement of antigen binding activityand of binding inhibition activity. The result, as shown in FIGS. 25 and27, indicated that this version was weaker for both activities ascompared to the above version h or version p and was similar to that ofthe above-mentioned a at most, suggesting that substitution of the aminoacid at position 78 is essential for the increase in the activity of thereshaped human antibody.

Version r of the H chain of the reshaped human anti-HM1.24 antibody wereevaluated by the method mentioned above. The result, as shown in FIGS.15 and 28, indicated that version r has a similar level of antigenbinding activity and the binding inhibition activity to that of thechimera anti-HM1.24 antibody.

The above results indicated that the minimum conversion required for thereshaped human anti-HM1.24 antibody to have a similar level of antigenbinding activity to that of the mouse anti-HM1.24 antibody or thechimera anti-HM1.24 antibody is the amino acids at positions 30, 71, and78 and, furthermore, 73.

The antigen binding activity and the binding inhibition activity for Hchain versions a to r of the reshaped human anti-HM1.24 antibody aresummarized in Table 2.

TABLE 2 Antigen binding Binding inhibition H chain version activityactivity a + + b + + c + + d + not measured e + not measured f ++ ++g + + h ++ ++ i ++ ++ j ++ ++ k ++ ++ l ++ ++ m ++ ++ n ++ ++ o ++ ++ p++ ++ q + + r +++ +++

Furthermore, the amino acid sequences of the reshaped human anti-HM1.24antibody and versions a and b of the L chain are shown in Table 3, andthose of versions a to r of the H chain of the reshaped humananti-HM1.24 antibody are shown in Tables 4 to 6.

TABLE 3 The amino acid sequence of the L chain V region  FR1                     CDR1        FR2         1         2          3          4 1234567890123456789012345678901234 56789012 AHM DIVMTQSHKFMSTSVGDRVSITC KASQDVNTAVA WYQQKPGQHuSG I DIQMTQSPSSLSASVGDRVTITC             WYQQKPGK REIDIQMTQSPSSLSASVGDRVTITC             WYQQKPGK RVLa----------------------- ----------- -------- RVLb----------------------- ----------- -------- 3456789 SPKLLIY APKLLIYAPKLLIY ------- -------  CDR2     FR3 5          6         7         80123456 78901234567890123456789012345678 AHM SASNRYTGVPDRITGSGSGTDFTFTISSVQAEDLALYYC HuSG I        GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC REI        GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC RVLa --------------------------------------- RVLb ---------------------Y-----------------   CDR3      FR4  9         10 9012345678901234567 AHM QQHYSTPFT FGSGTKLEIK HuSG I           FGQGTKVEIK REI          FGQGTKVEIK RVLa --------- ---------- RVLb --------- ----------

TABLE 4 The amino acid sequence of the H chain V region (1)  FR1                           CDR1   FR2         1         2         3           4123456789012345678901234567890 12345 6789012 3456789 AHMQVQLQQSGAELARPGASVKLSCKASGYTFT PYWMQ WVKQRPG QGLEWIG HuSGIEVQLVQSGADVKKPGXSVXVSCKASGYTFS       WVRQAPG XGLDWVG HG3QVQLVQSGAEVKKPGASVKVSCKASGYTFN       WVRQAPG QGLEWMG RVHa-----------------------------T ----- ------- ------- RVHb-----------------------------T ----- ------- ------- RVHc-----------------------------T ----- ------- ------- RVHd-----------------------------T ----- ------- ------- RVHe-----------------------------T ----- ------- ------- RVHf-----------------------------T ----- ------- ------- RVHg-----------------------------T ----- ----R-- ------- RVHh-----------------------------T ----- ------- ------- RVHi-----------------------------T ----- ------- ------- RVHj-----------------------------T ----- ------- ------- RVHk-----------------------------T ----- ------- ------- RVHl-----------------------------T ----- ------- ------- RVHm-----------------------------T ----- ------- ------- RVHn-----------------------------T ----- ------- ------- RVHo-----------------------------T ----- ------- ------- RVHp-----------------------------T ----- ------- ------- RVHq-----------------------------T ----- ------- ------- RVHr-----------------------------T ----- ------- -------

TABLE 5 The amino acid sequence of the H chain V region (2)  CDR2              FR3 5          6          7         8            9012A3456789012345 67890123456789012ABC345678901234 AHM SIFPGDGDTRYSQKFKGKATLTADKSSSTAYMQLSILAFEDSAVYYCAR HuSGI                  RVTXTXDXSXNTAYMELSSLRSEDTAVYYCAR HG3                  RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR RVHa----------------- -----A-------------------------- RVHb----------------- K----A-------------------------- RVHc----------------- -----A-K------------------------ RVHd----------------- K----A-K------------------------ RVHe----------------- -A-L-A-------------------------- RVHf----------------- -A-L-A---S--A------------------- RVHg----------------- -----A-------------------------- RVHh----------------- K----A---S--A------------------- RVHi----------------- K----A---S--A-------AF---------- RVHj----------------- KA-L-A---S--A------------------- RVHk----------------- K----A---S--A--Q---------------- RVHl----------------- K----A---S--A--Q--I------------- RVHm----------------- K----A---S--A--Q--I-----S------- RVHn----------------- K----A---S--A-----I------------- RVHo----------------- K----A---S--A-----------S------- RVHp----------------- -----A------A------------------- RVHq----------------- -----A---S---------------------- RVHr----------------- -----A-K----A-------------------

TABLE 6 The amino acid sequence of the H chain V region  CDR3        FR4   10             11 57890ABJK12  34567890123 AHMGLRRGGYYFDY  WGQGTTLTVSS HuSGI              WGQGTLVTVSS JH6             WGQGTTVTVSS RVHa ----------- ------------ RVHb ----------------------- RVHc ----------- ------------ RVHd ----------- ------------RVHe ----------- ------------ RVHf ----------- ------------ RVHg----------- ------------ RVHh ----------- ------------ RVHi ----------------------- RVHj ----------- ------------ RVHk ----------- ------------RVHl ----------- ------------ RVHm ----------- ------------ RVHn----------- ------------ RVHo ----------- ------------ RVHp ----------------------- RVHq ----------- ------------ RVHr ----------- ------------

3. Evaluation of the Purified Reshaped Human Anti-HM1.24 Antibody

The purified reshaped human anti-HM1.24 antibody was evaluated for theabove-mentioned antigen binding activity and binding inhibitionactivity. The result, as shown in FIGS. 31 and 32, indicated that thereshaped human anti-HM1.24 antibody has a similar level of antigenbinding activity and binding inhibition activity to that of the chimeraanti-HM1.24 antibody. This fact indicated that the reshaped humananti-HM1.24 antibody has the same antigen binding activity as the mouseanti-HM1.24 antibody.

Reference Example 12 Construction of the Hybridoma that Produces theMouse Anti-HM1.24 Monoclonal Antibody

The hybridoma that produces the mouse anti-HM1.24 monoclonal antibodywas prepared according to the method described in Goto, T. et al., Blood(1994) 84, 1992–1930.

The Epstein-Barr virus nuclear antigen (EBNA)-negative plasma cell lineKPC-32 (1×10⁷ cells) derived from the bone marrow of human patients withmultiple myeloma (Goto, T. et al., Jpn. J. Clin. Hematol. (11991) 32,1400) was intraperitoneally given twice to BALB/c mice (manufactured byCharles River) every six weeks.

In order to further elevate the titer of antibody production, 1.5×10⁶KPC-32 cells were injected into the spleen of the mice three days beforesacrificing the animals (Goto, T. et al., Tokushima J. Exp. Med. (1990)37, 89). After sacrificing the mice, the spleen was removed, and thespleen cells removed according to the method of Groth, de St. &Schreidegger (Cancer Research (1981) 41, 3465) were subjected to cellfusion with the myeloma cells SP2/0.

Antibody in the supernatant of the hybridoma culture was screened by theELISA (Posner, M. R. et al., J. Immunol. Methods (1982) 48, 23) usingthe KPC-32 cell-coated plates. 5×10⁴ KPC-32 cells were suspended in 50ml of PBS and dispensed into 96-well plates (U-bottomed, Corning,manufactured by Iwaki). After blocking with PBS containing 1% bovineserum albumin (BSA), the supernatant of the hybridoma was added andincubated at 4° C. for 2 hours. Subsequently, peroxidase-labeledanti-mouse IgG goat antibody (manufactured by Zymed) was reacted at 4°C. for 1 hour, washed once, and was reacted with the o-phenylenediaminesubstrate solution (manufactured by Sumitomo Bakelite) at roomtemperature for 30 minutes.

After stopping the reaction with 2N sulfuric acid, absorbance at 492 nmwas measured using the ELISA reader (manufactured by Bio-Rad). In orderto remove the hybridoma that produces antibody against humanimmunoglobulin, the positive hybridoma culture supernatant hadpreviously been adsorbed to human serum, and the reactivity to othersub-cellular components was screened. Positive hybridomas were selectedand their reactivity to various cell lines and human samples wasinvestigated using flow cytometry. The finally selected hybridoma cloneswere cloned twice, were injected into the abdominal cavity of thepristane-treated BALB/c mice and then the ascitic fluid was obtainedtherefrom.

Monoclonal antibody was purified from the mouse ascites by ammoniumsulfate precipitation and Protein A affinity chromatography kit (AmpurePa., manufactured by Amersham). The purified antibody was conjugated tofluorescein isocyanate (FITC) using the Quick Tag FITC conjugation kit(manufactured by Boehringer Mannheim).

As a result, the monoclonal antibody produced by 30 hybridoma clonesreacted with KPC-32 and RPMI 8226 cells. After cloning, the reactivityof the supernatant of these hybridomas with other cell lines andperipheral blood-derived monocytes was investigated.

Of them, three clones were monoclonal antibodies that specifically reactwith plasma cells. Out of these three clones, the hybridoma clone havingthe clone that is most useful for flow cytometry analysis and that hascomplement-dependent cytotoxicity was selected and termed HM1.24. Thesubclass of monoclonal antibody produced by this hybridoma wasdetermined by ELISA using subclass-specific anti-mouse rabbit antibody(manufactured by Zymed). Anti-HM1.24 antibody had a subclass of IgG2a κ.The hybridoma that produces the anti-HM1.24 antibody was internationallydeposited on Sep. 14, 1995, with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba city, Ibaraki prefecture, Japan) under theaccession number FERM BP-5233 under the provisions of the BudapestTreaty.

Reference Example 13 Cloning of cDNA Encoding the HM1.24 AntigenPolypeptide

1. Construction of cDNA Library

1) Preparation of Total RNA

The cDNA that encodes the HM1.24 antigen which is an antigen polypeptidespecifically recognized by mouse monoclonal antibody HM1.24 was isolatedas follows.

From the human multiple myeloma cell line KPMM2, total RNA was preparedaccording to the method of Chirgwin et al. (Biochemistry, 18, 5294(1979)). Thus, 2.2×10⁸ KPMM2 cells were completely homogenized in 20 mlof 4 M guanidine isocyanate (manufactured by Nacalai Tesque Inc.).

The homogenate was layered on the 5.3 M cesium chloride layer in thecentrifuge tube, which was then centrifuged using Beckman SW40 rotor at31,000 rpm at 20° C. for 24 hours to precipitate RNA. The RNAprecipitate was washed with 70% ethanol, and dissolved in 300 μl of 10mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 0.5% SDS. After addingPronase (manufactured by Boehringer) thereto to a concentration of 0.5mg/ml, it was incubated at 37° C. for 30 minutes. The mixture wasextracted with phenol and chloroform to precipitate RNA. Then, the RNAprecipitate was dissolved in 200 μl of 10 mM Tris-HCl (pH 7.4)containing 1 mM EDTA.

2) Preparation of Poly(A)+RNA

Using about 500 μg of the total RNA prepared as above as a raw material,poly(A)+RNA was purified using the Fast Track 2.0 m RNA Isolation Kit(manufactured by Invitrogen) according to the instructions attached tothe kit.

3) Construction of cDNA Library

Using 10 μg of the above poly(A)+RNA as a raw material, double strandcDNA was synthesized using the cDNA synthesizing kit TimeSaver cDNASynthesis Kit (manufactured by Pharmacia) according to the instructionsattached to the kit and, using the Directional Cloning Toolbox(manufactured by Pharmacia), EcoRI adapter was linked thereto accordingto the instructions attached to the kit. Kination and restriction enzymeNotI treatment of the EcoRI adapter were carried out according to theinstructions attached to the kit. Furthermore, the adapter-attacheddouble strand cDNA having a size of about 500 bp or higher was isolatedand purified using 1.5% agarose gel (manufactured by SIGMA) to obtainabout 40 μl of adapter-attached double strand cDNA.

The adapter-attached double strand cDNA thus prepared was linked usingpCOS1 vector (Japanese Unexamined Patent Publication (Kokai) No.8(1996)-255196) and T4 DNA ligase (manufactured by GIBCO BRL) that hadpreviously been treated with restriction enzymes EcoRI and NotI andalkaline phosphatase (manufactured by Takara Shuzo) to construct a cDNAlibrary. The constructed cDNA library was transduced into Escherichiacoli strain DH5 (manufactured by GIBCO BRL) and the total size wasestimated to be about 2.5×10⁶ independent cells.

2. Cloning by Direct Expression

1) Transfection into COS-7 Cells

cDNA was amplified by culturing about 5×10⁵ clones of the abovetransduced Escherichia coli in the 2-YT medium (Molecular Cloning: ALaboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press,(1989)) containing 50 μg/ml of ampicillin, and plasmid DNA was recoveredfrom the Escherichia coli by the alkali method (Molecular Cloning: ALaboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press,(1989)). The plasmid DNA obtained was transfected into COS-7 cells byelectroporation using the Gene Pulser instrument (manufactured byBioRad).

Thus, 10 μg of the purified plasmid DNA was added to 0.8 ml of COS-7cells that were suspended into PBS at a concentration of 1×10⁷ cells/ml,and was subjected to pulses at 1500 V and a capacity of 25 μF. After 10minutes of recovery period at room temperature, the electroporated cellswere cultured in the DMEM medium (manufactured by GIBCO BRL)supplemented with 10% bovine fetal serum under the condition of 37° C.and 5% CO₂ for three days.

2) Preparation of the Panning Dish

A panning dish coated with the mouse anti-HM1.24 antibody was preparedby the method of B. Seed et al. (Proc. Natl. Acad. Sci. USA, 84,3365–3369 (1987)). Thus, the mouse anti-HM1.24 antibody was added to 50mM Tris-HCl, pH 9.5, to a concentration of 10 μg/ml. Three ml of theantibody solution thus prepared was added to a tissue culture plate witha diameter of 60 mm and incubated at room temperature for 2 hours. Afterwashing three times with PBS containing 0.15 M NaCl, 5% bovine fetalserum, 1 mM EDTA, and 0.02% NaN₃ was added, and after blocking, it wasused for the following cloning.

3) Cloning of cDNA Library

The COS-7 cells transfected as described above were detached by PBScontaining 5 mM EDTA, and then washed once with PBS containing 5% bovinefetal serum. It was then suspended in PBS containing 5% bovine fetalserum and 0.02% NaN₃ to a concentration of about 1×10⁶ cells/ml, whichwas added to the panning dish prepared as above and incubated at roomtemperature for 2 hours. After washing three times with PBS containing5% bovine fetal serum and 0.02% NaN₃, plasmid DNA was recovered from thecells bound to the panning dish using a solution containing 0.6% SDS and10 mM EDTA.

The recovered plasmid DNA was transduced again to Escherichia coli DH5α.After amplifying the plasmid DNA as above, it was recovered by thealkali method. The recovered plasmid DNA was transfected into COS-7cells by the electroporation method to recover plasmid DNA from thebound cells as described above. The same procedure was repeated one moretime, and the recovered plasmid DNA was digested with restrictionenzymes EcoRI and NotI. As a result, concentration of the insert with asize of about 0.9 kbp was confirmed. Fifty μg of Escherichia colitransduced with part of the recovered plasmid DNA was inoculated to the2-YT agar plate containing 50 μg/ml of ampicillin. After culturingovernight, plasmid DNA containing a single colony was recovered. It wasdigested with restriction enzymes EcoRI and NotI and clone p3.19 havingan insert of 0.9 kbp was obtained.

The base sequence of this clone was determined by reacting using PRISM,Terminater Cycle Sequencing kit (manufactured by Perkin Elmer) accordingto the instructions attached to the kit. The amino acid sequence and thebase sequence thereof are shown in SEQ ID NO: 128.

The cDNA encoding the polypeptide having the amino acid sequence as setforth in SEQ ID NO: 128 was inserted into the XbaI cleavage site ofpUC19 vector, and has been prepared as plasmid pRS38-pUC19. TheEscherichia coli that contains this plasmid pRS38-pUC19 has beeninternationally deposited on Oct. 5, 1993, as Escherichia coli DH5α(pRS38-pUC19), with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba city, Ibaraki prefecture, Japan) under theaccession number FERM BP-4434 under the provisions of the BudapestTreaty (see Japanese Unexamined Patent Publication (Kokai) No.7(1995)-196694).

EXAMPLES

As an example of natural humanized antibodies composed of the natural FRsequences of the present invention, a preparation example of a naturalhumanized antibody based on humanized anti-HM1.24 antibody is described.

Example 1

Mouse monoclonal anti-HM1.24 antibody was humanized as the reshapedhuman anti-HM1.24 antibody by CDR-grafting as described in ReferenceExamples. Each FR of human antibody HG3 for FR1 to FR3 and the FR4 ofhuman antibody JH6 for FR4 were selected for the construction of thehumanized H chain. The result on the study of the FR amino acid residuesindicated that amino acid substitution was required at four sites(FR1/30, FR3/71, 73, 78) (Tables 7 and 8). This humanized antibody hadan antigen binding activity similar to that of the original antibody.This humanized antibody (humanized antibody comprising RVLa/RVHr) wasused as the primary design antibody.

Design of V region of Natural Humanized Antibody A) L chain  FR1                     CDR1        FR2            CDR2         1         2          3          4          512345678901234567890123 45678901234 567890123456789 0123456 HM1.24DIVMTQSHKFMSTSVGDRVSITC KASQDVNTAVA WYQQKPGQSPKLLIY SASNRYT HUSG IDIQMTQSPSSLSASVGDRVTITC             WYQQKPGKAPKLLIY REIDIQMTQSPSSLSASVGDRVTITC             WYQQKPGKAPKLLIY Primary design (RVLa----------------------- ----------- --------------- ------- Secondarydesign ----------------------- ----------- --------------- -------  FR3                              CDR3      FR4   6         7         8          9         1078901234567890123456789012345678 901234567 8901234567 HM1.24GVPDRITGSGSGTDFTFTISSVQAEDLALYYC QQHYSTPFT FGSGTKLEIK HUSG IGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC           FGQGTKVEIK REIGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC           FGQGTKVEIK Primary design(RVLa) -------------------------------- --------- ---------- Secondarydesign -------------------------------- --------- ----------

Design of V region of Natural Humanized Antibody B) H chain FR1                           CDR1   FR2            CDR2         1         2         3           4          5          6123456789012345678901234567890 12345 67890123456789 012A3456789012345HM1.24 QVQLQQSGAELARPGASVKLSCKASGYTFT PYWMQ WVKQRPGQGLEWIGSIFPGDGDTRYSQKFKG HuSGIEVQLVQSGADVKKPGXSVXVSCKASGYTFS       WVRQAPGXGLDWVG HG3QVQLVQSGAEVKKPGASVKVSCKASGYTFN       WVRQAPGQGLEWMG Primary design(RVHr) -----------------------------T ----- ------------------------------- Secondary design (2ndRVH)-----------------------------T ----- -------------- ----------------- FR3                             CDR3          FR4    7         8            9        10             1167890123456789012ABC345678901234 57890ABJK12 34567890123 HM1.24KATLTADKSSSTAYMQLSILAFEDSAVYYCAR GLRRGGYYFDY WGQGTTLTVSS HuSGIRVTXTXDXSXNTAYMELSSLRSEDTAVYYCAR             WGQGTLVTVSS HG3 JH6RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR             WGQGTTVTVSS Primary design(RVHr) -----A-K----A------------------- ----------- -----------Secondary design (2ndRVH) ---I-A-K----A------------------- ----------------------

(1) The Construction of H Chain

For the FR of the primary design antibody, homology search on human FRsfound in nature was carried out using such databases as SeissPlot,GenBank, PRF, PIR, and GenPept. First, 50 human FRs were found that havecompletely matching amino acid sequences for FR1. Thus, the FR1 of theprimary design antibody already had a natural sequence. Since no aminoacid substitution has been made for FR2 and FR4, 50 and 100 natural FRsincluding HG3 and JH6 respectively of natural human body were found.

On the other hand, no complete matches were found for FR3. As the FR3that had the highest homology, S46463 having a homology of 96.875%,1921296C, HUMIGHRF 1, U00583 1 and the like were found (symbols are allaccession numbers for the database).

Thus, in the primary design antibody, FR3 was the FR containingartificial amino acid residues that are not found in nature. The aminoacid sequence is compared with that of the human antibody S46463 thathad the highest homology in Table 9.

FR3 of primary design antibody        |       |                                     10        20        30       →                             RYTMTADKSTSTAYMELSSLRSEDTAVYYCAR                                     ... ............................                                     ................................       →VRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR       |  40        50        60        70        80        90        |FR3 of S46463 antibody

The amino acid residue at position 70 was methionine in the FR3 of theprimary design antibody and was isoleucine in the FR3 of the humanantibody S46463. The other amino acid sequences have shown completematches. Thus, the amino acid residue at position 70 in the primarydesign antibody was replaced with isoleucine to convert it to anaturally occurring FR3. Accordingly, the secondary design antibodyobtained is a CDR-grafting antibody comprising the natural human FR ofthe human antibody S46463. The secondary design antibody thusconstructed comprises FRs that are all found in nature.

(2) Construction of the H Chain V Region of Natural HumanizedAnti-HM1.24 Antibody

The H chain V region of the natural humanized anti-HM 1.24 antibody wasconstructed by mutagenesis using PCR. The mutagen primers SS (SEQ ID NO:124) and SA (SEQ ID NO: 125) were designed to mutate methionine atposition 69 to isoleucine.

After the above primer was amplified using plasmid HEF-RVHr-AHM-gγ1 as atemplate, the final product was purified, digested with BamHI andHindIII, and the DNA fragment obtained was cloned into an expressionvector HEF-VH-gγ1 to obtain a plasmid HEF-RVHs-AHM-gγ1. The amino acidsequence and the nucleotide sequence of the V region of the H chaincontained in this plasmid HEF-RVHs-AHM-gγ1 are shown in SEQ ID NO: 126.

The region encoding the variable region of the above-mentioned plasmidHEF-RVHs-AHM-gγ1 was digested with restriction enzymes HindIII and BamHIto make a restriction fragment. This was inserted into the BamHI andHindIII sites of plasmid vector pUC19. The plasmid obtained was termedpUC19-RVHs-AHM-gγ1.

Escherichia coli that contains pUC19-RVHs-AHM-gγ1 was designated asEscherichia coli DH5α (pUC19-RVHs-AHM-gγ1) and has been internationallydeposited on Sep. 29, 1997, with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba city, Ibaraki prefecture, Japan) under theaccession number FERM BP-6127 under the provisions of the BudapestTreaty.

2) Analysis of L Chain

Although amino acids of the FRs were not substituted in the constructionof the L chain of the primary design antibody, homology search wasconducted also for these FRs, since the human antibody REI used was aReshaped FR (Riechmann, L. et al., Nature (1988) 332, 323–327) that hadalready been subjected to amino acid substitution. The result confirmedthe presence of natural sequences corresponding to the reshaped FRs.Thus, it was demonstrated that no amino acid substitution is requiredfor FRs of L chain.

Example 2 Production of Natural Humanized Anti-HM1.24 Antibody

(1) Expression of Natural Humanized Anti-HM1.24 Antibody

Ten μg each of the expression vector (HEF-RVHs-AHM-gγ1) for H chain ofnatural humanized anti-HM1.24 antibody and the expression vector(HEF-RVLa-AHM-gκ) for L chain of reshaped human anti-HM1.24 antibody wascotransformed into COS cells by electroporation using the Gene Pulserinstrument (manufactured by BioRad). Each DNA (10 μg) was added to 0.8ml aliquots of 1×10⁷ cells/ml in PBS, and was subjected to pulses at1500 V and a capacity of 25 μF.

After a recovery period of 10 minutes at room temperature, theelectroporated cells were added to 30 ml of DHEM culture liquid(manufactured by GIBCO) containing 10% γ-globulin-free bovine fetalserum. After incubation of 72 hours in a CO₂-incubator BNA120D(manufactured by TABAI) under the condition of 37° C. and 5% CO₂, theculture supernatant was collected, and the cell debris was removed bycentrifugation at 1000 rpm for 5 minutes in a centrifuge 505PR-22(manufactured by HITACHI) equipped with a centrifuge rotor 03(manufactured by HITACHI). Then ultrafiltration was carried out with amicroconcentrator (Centricon 100, manufactured by Amicon) using acentrifuge J2-21 (manufactured by BECKMAN) equipped with a centrifugerotor JA-20.1 (manufactured by BECKMAN), at a condition of 2000 rpm, andfilter-sterilization was carried out using a filter Milex GV13 mm(manufactured by Millipore) to obtain a product which was used forCell-ELISA.

(2) Measurement of Antibody Concentration

Concentration of the antibody obtained was measured by ELISA. To eachwell of a 96-well ELISA plate (Maxisorp, manufactured by NUNC) was added100 μl of goat anti-human IgG antibody (manufactured by BIO SOURCE)prepared to a concentration of 1 μg/ml with the coating buffer (0.1 MNaHCO₃, 0.02% NaN₃, pH 9.6) and the plate was incubated at roomtemperature for one hour. After blocking with 100 μl of the dilutionbuffer (50 mM Tris-HCl, 1 MM MgCl₂, 0.15 M NaCl, 0.05% Tween 20, 0.02%NaN₃, 1% bovine serum albumin (BSA), pH 8.1), 100 μl each of serialdilutions of the natural humanized anti-HM1.24 antibody was added toeach well and the plate was incubated at room temperature for one hour.Then after washing, 100 μl of alkaline phosphatase-labeled goatanti-human IgG antibody (manufactured by DAKO) was added.

After incubating at room temperature for one hour and washing, 100 μl of1 mg/ml substrate solution (Sigma 104, p-nitrophenyl phosphate,manufactured by SIGMA) dissolved in substrate buffer (50 mM NaHCO₃, 10mM MgCl₂, pH 9.8) was added, and then the absorbance at 405 nm wasmeasured using the MICROPLATE READER Model 3550 (manufactured by BioRad). As a standard for measurement of concentration, human IgG1κ(manufactured by The Binding Site) was used.

(3) Establishment of the CHO Cell Line that Stably Produces the NaturalHumanized Anti-HM1.24 Antibody

The CHO cell line that stably produces the natural humanized anti-HM1.24antibody can be established according to the following method.

(3)-1. Construction of an Expression Vector for an H Chain of a NaturalHumanized Anti-HM1.24 Antibody

By digesting plasmid HEF-RVHs-AHM-gγ1 with restriction enzymes PvuI andBamHI, an about 2.8 kbp fragment containing DNA encoding an EF1 promoterand a V region of the H chain of natural humanized anti-HM1.24 antibodywas purified using 1.5% low melting point agarose gel. Then, the aboveDNA fragment is inserted into an about 6 kbp fragment that was preparedby digesting with PvuI and BamHI the expression vector used for a humanH chain expression vector, DHFR-ΔE-RVh-PM1f (International PatentPublication No. WO 92-19759), containing a DHFR gene and a gene encodinga constant region of a human H chain, so as to construct an expressionvector, DHFR-ΔE-HEF-RVHs-AHM-gγ1, for the H chain of the naturalhumanized anti-HM1.24 antibody.

(3)-2. Gene Introduction into CHO Cells

In order to establish a stable production system of the naturalhumanized anti-HM1.24 antibody, the genes of the above-mentionedexpression vectors, DHFR-ΔE-RVHs-AHM-gγl and HEF-RVLa-AHM-gκ, that werelinearized by digestion with PvuI, were simultaneously introduced intothe CHO cell DXB-11 by the electroporation method under the conditionsimilar to the above-mentioned one (transfection into theabove-mentioned COS-7 cells).

(3)-3. Gene Amplification by MTX

Of the gene-introduced CHO cells, only those CHO cells in which both ofL chain and H chain expression vectors have been introduced can survivein the nucleoside-free α-MEM culture liquid (manufactured by GIBCO-BRL)to which 500 μg/ml G418 (manufactured by GIBCO-BRL) and 10% bovine fetalserum were added, and so they were selected. Subsequently, 10 nM MTX(manufactured by Sigma) is added to the above culture. Of the clonesthat propagated, those that produce a natural humanized anti-HM1.24antibody in large amount were selected.

(3)-4. Construction of the Natural Humanized Anti-HM1.24 Antibody

The natural humanized anti-HM1.24 antibody was produced in the followingmethod. The above CHO cells that produce the natural humanizedanti-HM1.24 antibody were cultured for 10 days using a nucleoside-freeα-MEM culture medium (manufactured by GIBCO-BRL) to which 500 μg/ml G418(manufactured by GIBCO-BRL) containing 10% γ-globulin-free bovine fetalserum (manufactured by GIBCO-BRL) had been added, using a CO₂ incubatorBNAS120D (manufactured by TABAI) under the condition of 37° C. and 5%CO₂. On day 8 and 10 after starting the culture the culture medium wasrecovered, the cell debris was removed by centrifuging for 10 minutes at2000 rpm using the centrifuge RL-500SP (manufactured by Tomy Seiko)equipped with the TS-9 rotor, and then filter-sterilized using a bottletop filter (manufactured by FALCON) having a membrane with pores of 0.45μm in diameter.

After an equal amount of PBS(−) was added to the culture liquid of theCHO cells that produce the natural humanized anti-HM1.24 antibody, thenthe natural humanized anti-HM1.24 antibody was affinity-purified usingthe high-speed antibody purification system ConSep LC100 (manufacturedby MILLIPORE) and Hyper D Protein A column (manufactured by NipponGaishi) using PBS(−) as an absorption buffer and 0.1 M sodium citratebuffer (pH 3) as an elution buffer, according to the attachedinstructions. The eluted fractions were adjusted to about pH 7.4 byimmediately adding 1 M Tris-HCl (pH 8.0) and then using the centrifugingultrafiltration concentrator Centriprep 10 (manufactured by MILLIPORE),concentration and substitution to PBS(−) were carried out and theproduct was filter-sterilized using a membrane filter MILLEX-GV(manufactured by MILLIPORE) with a pore size of 0.22 μm to obtain thepurified natural humanized anti-HM1.24 antibody. Concentration ofpurified antibody was measured by absorbance at 280 nm and calculated as1 μg/ml per 1.35 OD.

Example 3 Determination of Activity of the Natural Humanized Anti-HM1.24Antibody

The natural humanized anti-HM1.24 antibody was evaluated for thefollowing antigen binding activity, binding inhibition activity, andADCC activity.

(1) The Method of Measurement of Antigen Binding Activity and BindingInhibition Activity

(1)-1. Measurement of Antigen Binding Activity

Antigen binding activity was measured by Cell-ELISA using WICH cells.Cell-ELISA plates were prepared as described in the above ReferenceExample 7.1-2.

After blocking, 100 μl of serial dilutions of the natural humanizedanti-HM1.24 antibody that was obtained from a concentrate of a culturesupernatant of COS-7 cells was added to each well. After it wasincubated for 2 hours at room temperature and washed, peroxidase-labeledrabbit anti-human IgG antibody (manufactured by DAKO) was added. Afterincubating for 2 hours at room temperature and washing, a substratesolution was added and incubated. Then the reaction was stopped byadding 50 μl of 6N sulfuric acid, and absorbance at 490 nm was measuredusing the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

(1)-2. Measurement of Binding Inhibition Activity

The binding inhibition activity by the biotin-labeled mouse anti-HM1.24antibody was measured by the Cell-ELISA using WISH cells. Cell-ELISAplates were prepared as described above. After blocking, 50 μl of serialdilutions of the natural humanized anti-HM1.24 antibody that wasobtained from the concentrate of the culture supernatant of COS-7 cellswas added to each well, and 50 μl of 2 μg/ml biotin-labeled mouseanti-HM1.24 antibody was added simultaneously. After incubating at roomtemperature for two hours and washing, peroxidase-labeled streptoavidin(manufactured by DAKO) was added. After incubating at room temperaturefor one hour and washing, the reaction was stopped by adding 50 μl of 6Nsulfuric acid, and absorbance at 490 nm was measured using theMICROPLATE READER Model 3550 (manufactured by Bio-Rad).

(2) Antigen Binding Activity and Binding Inhibition Activity

The evaluation of the H chain of natural humanized anti-HM1.24 antibodywas conducted by measurement of the above-mentioned antigen bindingactivity and binding inhibition activity in combination with the L chainversion a. The result, as shown in FIGS. 29 and 30, indicated thatnatural humanized anti-HM1.24 antibody (the secondary design antibody)has antigen binding activity and binding inhibition activity of asimilar degree to the primary design antibody (reshaped humananti-HM1.24 antibody: the H chain version r).

(3) Measurement of the ADCC Activity

ADCC (Antibody-dependent Cellular Cytotoxicity) activity was measuredaccording to the method described in Reference Example 8.

1. Preparation of Effector Cells

To the peripheral blood of healthy human subject was added an equalamount of PBS(−), onto which Ficoll-Paque (manufactured by Pharmacia)was layered, and was centrifuged at 500 g for 30 minutes. The monocytelayer was taken therefrom and was washed twice with RPMI 1640(manufactured by GIBCO BRL) supplemented with 10% bovine fetal serum(manufactured by GIBCO BRL), and was adjusted to a cell density of5×10⁶/ml with the same culture liquid.

2. Preparation of Target Cells

The human myeloma cell line KPMM2 (Deposit No. P-14170, Patentapplication No. 6-58082) was radiolabeled by incubating in RPMI 1640(manufactured by GIBCO BRL) supplemented with 10% bovine fetal serum(manufactured by GIBCO BRL) together with 0.1 mCi of ⁵¹Cr-sodiumchromate at 37° C. for 60 minutes. After radiolabeling, cells werewashed three times with the same buffer and adjusted to a concentrationof 2×10⁵/ml.

3. Measurement of ADCC Assay

Into a 96-well U-bottomed plate (manufactured by Corning) were added 50μl of 2×10⁵ target cells/ml, 50 μl of the antibody solution previouslyprepared at 4 μg/ml, 0.4 μg/ml, 0.04 μg/ml, and 0.004 μg/ml, and reactedat 4° C. for 15 minutes. A solution that does not contain naturalhumanized anti-HM1.24 antibody (the secondary design antibody) wassimilarly prepared and used as a control.

Then, 100 μl of 5×10⁵ effector cells/ml was added thereto, and culturedin a CO₂-incubator for 4 hours, wherein the ratio (E:T) of the effectorcells (E) to the target cells (T) was set at 0:1, 20:1, and 50:1. Sincethe final concentration of each antibody was diluted by four-fold, theywere 1 μg/ml, 0.1 μg/ml, 0.01 μg/ml, and 0.001 μg/ml as well as noantibody addition control.

One hundred μl of the supernatant was taken and the radioactivityreleased into the culture supernatant was measured by a gamma counter(ARC361, manufactured by Aloka). For measurement of the maximumradioactivity, 1% NP-40 (manufactured by Nacalai Tesque Inc.) was used.Cytotoxicity (%) was calculated by (A−C)/(B−C)×100, wherein A isradioactivity (cpm) released in the presence of antibody, B isradioactivity (cpm) released by NP-40, and C is radioactivity (cpm)released by the culture medium alone without antibody.

4. Result

As shown in FIG. 33, when the natural humanized anti-HM1.24 antibody(the secondary design antibody) was added, specific chromium releaserate increased with the increase in the E:T ratio depending on antibodyconcentration as compared to the no antibody added control. This,therefore, indicated that this natural humanized anti-HM1.24 antibody(the secondary design antibody) has ADCC activity.

The present invention relates to a method of preparing natural humanizedantibody and the natural humanized antibody obtained by said method ofpreparation. This is a highly excellent humanization technology that hassolved the problems associated with CDR-grafting (Jones, P. T. et al.,Nature (1986) 321, 522–525) created by G. Winter. Construction of theprimary design antibody may be considered as an intermediate stage forthe construction of humanized antibody comprising natural human FRs.When antibody is developed as a pharmaceutical product comprisingrecombinant protein, natural humanized antibody that comprises naturallyoccurring human FRs is more excellent in terms of antigenicity andsafety.

Effects of the Invention

Since the natural humanized antibody obtained by the method ofpreparation of the present invention does not contain the amino acidresidues of non-naturally occurring artificial FRs that are contained inthe humanized antibody produced by the conventional humanizzationtechnology, it is expected to have low antigenicity. Furthermore, it wasshown that the natural humanized antibody obtained by the method ofpreparation of the present invention has an activity similar to that ofantibody derived from a non-human mammal that was used as a template forhumanization. Therefore, the natural humanized antibody obtained by themethod of preparation of the present invention is useful for therapeuticadministration to humans.

Reference to the microorganisms deposited under the Patent CooperationTreaty, Rule 13-2, and the name of the Depository Institute

Depository Institute

Name: the National Institute of Bioscience and Human Technology, Agencyof Industrial Science and Technology

Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan

Organism (1)

-   Indication: Escherichia coli DH5α (pRS38-pUC19)-   Accession number: FERM BP-4434-   Deposition Date: Oct. 5, 1993

Organism (2)

-   Indication: Hybridoma HM1.24-   Accession number: FERM BP-5233-   Deposition Date: Sep. 14, 1995

Organism (3)

-   Indication: Escherichia coli DH5α (pUC19-RVHr-AHM-gγ1)-   Accession number: FERM BP-5643-   Deposition Date: Aug. 29, 1996

Organism (4)

-   Indication: Escherichia coli DH5α (pUC19-1.24H-gγ1)-   Accession number: FERM BP-5644-   Deposition Date: Aug. 29, 1996

Organism (5)

-   Indication: Escherichia coli DH5α (pUC19-RVLa-AHM-gκ)-   Accession number: FERM BP-5645-   Deposition Date: Aug. 29, 1996

Organism (6)

-   Indication: Escherichia coli DH5α (pUC19-RVHs-AHM-gγ1)-   Accession number: FERM BP-6127-   Deposition Date: Sep. 29, 1997

1. A method for preparing a humanized antibody, wherein a frameworkregion (“FR”) in the humanized antibody is a FR naturally occurring inhuman antibodies, comprising the steps of: (1) obtaining a humanizedantibody, wherein the humanized antibody has: i) six complementarydetermining regions (“CDRs”) of a first animal species; and ii) eightFRs of a second animal species, wherein one or more amino acid residuesin one or more of the FRs have been substituted to retain antigenbinding ability, with corresponding amino acid residues in FRs of thefirst animal species, and wherein said second animal species is human;(2) conducting a homology search using a database of amino acid sequenceof FRs naturally occurring in human antibodies (“natural FRs”) incomparison with the amino acid sequence of the humanized antibodyobtained in step (1), wherein the homology search is conducted over all8FRs; (3) preparing a list of amino acid sequences of the natural FRshaving the same as or at least 80% homology with the amino acid sequenceof the FR, of the humanized antibody obtained in step (1), (4)selecting, from the list of step (3), a natural FR which has i) atcorresponding positions the same amino acid residues as the amino acidresidues introduced by the substitution in step (1); and ii) comprisesan amino acid sequence that is the same as or has at least 80% homologywith the FR sequence of the humanized antibody obtained in step (1); (5)if the amino acid sequence of the FR, in which amino acid residues havebeen substituted in step (1), of the humanized antibody obtained in step(1) has one or more amino acid residues that are different from aminoacid residues at corresponding positions of the natural FR selected instep (4), replace said different amino acid residues in the FR sequenceof the humanized antibody obtained in step (1) with corresponding aminoacid residues in the natural FR; (6) constructing an expression vectorexpressing an amino acid sequence of the antibody obtained via steps (1)to (5); (7) culturing cells comprising an expression vector constructedin step (6); and (8) recovering from the culture the humanized antibodycomprising the natural FR and 6 CDRs from the first animal species andwherein the recovered humanized antibody binds the same antigen that theantibody from the first animal species binds.
 2. The method according toclaim 1, wherein the first animal species is a non-human mammal.
 3. Themethod according to claim 2, wherein the non-human mammal is selectedfrom a mouse, rat, hamster, rabbit and monkey.
 4. The method accordingto claim 1, wherein all natural FRs belong to the same subgroup.
 5. Themethod according to claim 1, wherein the number of the substituted aminoacid residues of the FR in step (1) is from one to ten.
 6. The methodaccording to claim 1, wherein the number of the different amino acidresidues in the FR in step (5) is from one to ten.
 7. The methodaccording to claim 1, wherein the substituted amino acid residues in theFR in step (1) comprise an amino acid residue selected from amino acidresidues responsible for canonical structure of the antibody, amino acidresidues involved in the maintenance of the structure of CDRs, and theamino acid residues that directly interact with an antigen.
 8. Themethod according to claim 5, wherein the substituted amino acid residuesin the FR in step (1) comprise an amino acid residue selected from anamino acid residue at position 71 of the heavy chain or at position 94of the heavy chain.